Processes for producing polyunsaturated fatty acids in transgenic organisms

ABSTRACT

The present invention relates to polynucleotides from  Ostreococcus lucimarinus  which code for desaturases and elongases and which can be employed for the recombinant production of polyunsaturated fatty acids. The invention furthermore relates to vectors, host cells and transgenic nonhuman organisms which comprise the polynucleotides, and to the polypeptides encoded by the polynucleotides. Finally, the invention also relates to production processes for the polyunsaturated fatty acids and for oil, lipid and fatty acid compositions.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/911,675, filed Jun. 6, 2013, which is a divisional of U.S.application Ser. No. 12/444,193, filed Apr. 3, 2009, now U.S. Pat. No.8,710,299, which is a national stage application (under 35 U.S.C. §371)of PCT/EP2007/060554, filed Oct. 4, 2007, which claims benefit ofEuropean Application No. 06121888.9, filed Oct. 6, 2006. The entirecontents of each of these applications are hereby incorporated byreference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Sequence_Listing_074040_0113_01. The size of thetext file is 199 KB, and the text file was created on Jun. 6, 2016.

DESCRIPTION

The present invention relates to polynucleotides from Ostreococcuslucimarinus which code for desaturases and elongases and which can beemployed for the recombinant production of polyunsaturated fatty acids.The invention furthermore relates to vectors, host cells and transgenicnonhuman organisms which comprise the polynucleotides, and to thepolypeptides encoded by the polynucleotides. Finally, the invention alsorelates to production processes for the polyunsaturated fatty acids andfor oil, lipid and fatty acid compositions.

Fatty acids and triacylglycerides have a multiplicity of applications inthe food industry, in animal nutrition, in cosmetics and in thepharmacological sector. Depending on whether they are free saturated orunsaturated fatty acids or else triacylglycerides with an elevatedcontent of saturated or unsaturated fatty acids, they are suitable forvery different applications. Polyunsaturated fatty acids such aslinoleic acid and linolenic acid are essential for mammals, since theycannot be produced by the latter. Polyunsaturated ω3-fatty acids andω6-fatty acids are therefore an important constituent in animal andhuman nutrition.

Polyunsaturated long-chain ω3-fatty acids such as eicosapentaenoic acid(=EPA, C20:5^(Δ5,8,11,14,17)) or docosahexaenoic acid (=DHA,C22:6^(Δ4,7,10,13,16,19)) are important components in human nutritionowing to their various roles in health aspects, including thedevelopment of the child brain, the functionality of the eyes, thesynthesis of hormones and other signal substances, and the prevention ofcardiovascular disorders, cancer and diabetes (Poulos, A Lipids 30:1-14,1995; Horrocks, L A and Yeo Y K Pharmacol Res 40:211-225, 1999). This iswhy there is a demand for the production of polyunsaturated long-chainfatty acids.

Owing to the currently customary composition of human food, an additionof polyunsaturated ω3-fatty acids, which are preferentially found infish oils, to the food is particularly important. Thus, for example,polyunsaturated fatty acids such as docosahexaenoic acid (=DHA,C22:6^(Δ4,7,10,13,16,19)) or eicosapentaenoic acid (=EPA,C20:5^(Δ5,8,11,14,17)) are added to infant formula to improve thenutritional value. The unsaturated fatty acid DHA is said to have apositive effect on the development and maintenance of brain functions.

Hereinbelow, polyunsaturated fatty acids are referred to as PUFA, PUFAs,LCPUFA or LCPUFAs (poly unsaturated fatty acids, PUFA, long chain polyunsaturated fatty acids, LCPUFA).

The various fatty acids and triglycerides are mainly obtained frommicroorganisms such as Mortierella and Schizochytrium or fromoil-producing plants such as soybean, oilseed rape, algae such asCrypthecodinium or Phaeodactylum and others, where they are obtained, asa rule, in the form of their triacylglycerides(=triglycerides=triglycerols). However, they can also be obtained fromanimals, such as, for example, fish. The free fatty acids areadvantageously prepared by hydrolysis. Very long-chain polyunsaturatedfatty acids such as DHA, EPA, arachidonic acid (=ARA,C20:4^(Δ5,8,11,14)), dihomo-γ-linolenic acid (C20:3^(Δ8,11,14)) ordocosapentaenoic acid (DPA, C22:5^(Δ7,10,13,16,19)) are not synthesizedin oil crops such as oilseed rape, soybean, sunflower or safflower.Conventional natural sources of these fatty acids are fish such asherring, salmon, sardine, redfish, eel, carp, trout, halibut, mackerel,zander or tuna, or algae.

Depending on the intended use, oils with saturated or unsaturated fattyacids are preferred. In human nutrition, for example, lipids withunsaturated fatty acids, specifically polyunsaturated fatty acids, arepreferred. The polyunsaturated ω3-fatty acids are said to have apositive effect on the cholesterol level in the blood and thus on thepossibility of preventing heart disease. The risk of heart disease,stroke or hypertension can be reduced markedly by adding these ω3-fattyacids to the food. Also, ω3-fatty acids have a positive effect oninflammatory, specifically on chronically inflammatory, processes inassociation with immunological diseases such as rheumatoid arthritis.They are therefore added to foodstuffs, specifically to dieteticfoodstuffs, or are employed in medicaments. ω6-Fatty acids such asarachidonic acid tend to have a negative effect on these disorders inconnection with these rheumatic diseases on account of our usual dietaryintake.

ω3- and ω6-fatty acids are precursors of tissue hormones, known aseicosanoids, such as the prostaglandins, which are derived fromdihomo-γ-linolenic acid, arachidonic acid and eicosapentaenoic acid, andof the thromoxanes and leukotrienes, which are derived from arachidonicacid and eicosapentaenoic acid. Eicosanoids (known as the PG₂ series)which are formed from ω6-fatty acids generally promote inflammatoryreactions, while eicosanoids (known as the PG₃ series) from ω3-fattyacids have little or no proinflammatory effect.

Owing to the positive characteristics of the polyunsaturated fattyacids, there has been no lack of attempts in the past to make availablegenes which are involved in the synthesis of fatty acids ortriglycerides for the production of oils in various organisms with amodified content of unsaturated fatty acids. Thus, WO 91/13972 and itsUS equivalent describes a Δ9-desaturase. WO 93/11245 claims aΔ15-desaturase and WO 94/11516 a Δ12-desaturase. Further desaturases aredescribed, for example, in EP-A-0 550 162, WO 94/18337, WO 97/30582, WO97/21340, WO 95/18222, EP-A-0 794 250, Stukey et al., J. Biol. Chem.,265, 1990: 20144-20149, Wada et al., Nature 347, 1990: 200-203 or Huanget al., Lipids 34, 1999: 649-659. However, the biochemicalcharacterization of the various desaturases has been insufficient todate since the enzymes, being membrane-bound proteins, present greatdifficulty in their isolation and characterization (McKeon et al.,Methods in Enzymol. 71, 1981: 12141-12147, Wang et al., Plant Physiol.Biochem., 26, 1988: 777-792). As a rule, membrane-bound desaturases arecharacterized by being introduced into a suitable organism which issubsequently analyzed for enzyme activity by analyzing the startingmaterials and the products. Δ6-Desaturases are described in WO 93/06712,U.S. Pat. No. 5,614,393, WO 96/21022, WO 00/21557 and WO 99/27111 andthe application for the production in transgenic organisms is describedin WO 98/46763, WO 98/46764 and WO 98/46765. In this context, theexpression of various desaturases and the formation of polyunsaturatedfatty acids is also described and claimed in WO 99/64616 or WO 98/46776.As regards the expression efficacy of desaturases and its effect on theformation of polyunsaturated fatty acids, it must be noted that theexpression of a single desaturase as described to date has only resultedin low contents of unsaturated fatty acids/lipids such as, for example,γ-linolenic acid and stearidonic acid. Moreover, a mixture of ω3- andω6-fatty acids was obtained, as a rule.

Especially suitable microorganisms for the production of PUFAs aremicroalgae such as Phaeodactylum tricornutum, Porphiridium species,Thraustochytrium species, Schizochytrium species or Crypthecodiniumspecies, ciliates such as Stylonychia or Colpidium, fungi such asMortierella, Entomophthora or Mucor and/or mosses such asPhyscomitrella, Ceratodon and Marchantia (R. Vazhappilly & F. Chen(1998) Botanica Marina 41: 553-558; K. Totani & K. Oba (1987) Lipids 22:1060-1062; M. Akimoto et al. (1998) Appl. Biochemistry and Biotechnology73: 269-278). Strain selection has resulted in the development of anumber of mutant strains of the microorganisms in question which producea series of desirable compounds including PUFAs. However, the mutationand selection of strains with an improved production of a particularmolecule such as the polyunsaturated fatty acids is a time-consuming anddifficult process. This is why recombinant methods as described aboveare preferred whenever possible.

However, only limited amounts of the desired polyunsaturated fatty acidssuch as DPA, EPA or ARA can be produced with the aid of theabovementioned microorganisms, and, depending on the microorganism used,these are generally obtained as fatty acid mixtures of, for example,EPA, DPA and ARA.

A variety of synthetic pathways is being discussed for the synthesis ofarachidonic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid(DHA). Thus, EPA or DHA are produced in marine bacteria such as Vibriosp. or Shewanella sp. via the polyketide pathway (Yu, R. et al. Lipids35:1061-1064, 2000; Takeyama, H. et al. Microbiology 143:2725-2731,1997).

An alternative strategy is the alternating activity of desaturases andelongases (Zank, T. K. et al. Plant Journal 31:255-268, 2002;Sakuradani, E. et al. Gene 238:445-453, 1999). A modification of theabove-described pathway by Δ6-desaturase, Δ6-elongase, Δ5-desaturase,Δ5-elongase and Δ4-desaturase is the Sprecher pathway (Sprecher 2000,Biochim. Biophys. Acta 1486:219-231) in mammals. Instead of theΔ4-desaturation, a further elongation step is effected here to give C₂₄,followed by a further Δ6-desaturation and finally 1-oxidation to givethe C₂₂ chain length. Thus what is known as Sprecher pathway is,however, not suitable for the production in plants and microorganismssince the regulatory mechanisms are not known.

Depending on their desaturation pattern, the polyunsaturated fatty acidscan be divided into two large classes, viz. ω6- or ω3-fatty acids, whichdiffer with regard to their metabolic and functional activities.

The starting material for the ω6-metabolic pathway is the fatty acidlinoleic acid (18:2^(Δ9,12)) while the ω3-pathway proceeds via linolenicacid (18:3^(Δ9,12,15)). Linolenic acid is formed by the activity of anω3-desaturase (Tocher et al. 1998, Prog. Lipid Res. 37, 73-117; Domergueet al. 2002, Eur. J. Biochem. 269, 4105-4113).

Mammals, and thus also humans, have no corresponding desaturase activity(Δ12- and ω3-desaturase) and must take up these fatty acids (essentialfatty acids) via the food. Starting with these precursors, thephysiologically important polyunsaturated fatty acids arachidonic acid(=ARA, 20:4^(Δ5,8,11,14)), an ω6-fatty acid and the two ω3-fatty acidseicosapentaenoic acid (=EPA, 20:5^(Δ5,8,11,14,17)) and docosahexaenoicacid (DHA, 22:6^(Δ4,7,10,13,17,19)) are synthesized via the sequence ofdesaturase and elongase reactions. The application of ω3-fatty acidsshows the therapeutic activity described above in the treatment ofcardiovascular diseases (Shimikawa 2001, World Rev. Nutr. Diet. 88,100-108), inflammations (Calder 2002, Proc. Nutr. Soc. 61, 345-358) andarthritis (Cleland and James 2000, J. Rheumatol. 27, 2305-2307).

The elongation of fatty acids, by elongases, by 2 or 4 C atoms is ofcrucial importance for the production of C₂₀- and C₂₂-PUFAs,respectively. This process proceeds via 4 steps. The first step is thecondensation of malonyl-CoA onto the fatty acid-acyl-CoA by ketoacyl-CoAsynthase (KCS, hereinbelow referred to as elongase). This is followed bya reduction step (ketoacyl-CoA reductase, KCR), a dehydratation step(dehydratase) and a final reduction step (enoyl-CoA reductase). It hasbeen postulated that the elongase activity affects the specificity andrate of the entire process (Millar and Kunst, 1997 Plant Journal12:121-131).

There have been a large number of attempts in the past to obtainelongase genes. Millar and Kunst, 1997 (Plant Journal 12:121-131) andMillar et al. 1999, (Plant Cell 11:825-838) describe thecharacterization of plant elongases for the synthesis of monounsaturatedlong-chain fatty acids (C22:1) and for the synthesis of very long-chainfatty acids for the formation of waxes in plants (C₂₈-C₃₂). Descriptionsregarding the synthesis of arachidonic acid and EPA are found, forexample, in WO0159128, WO0012720, WO02077213 and WO0208401. Thesynthesis of polyunsaturated C₂₄-fatty acids is described, for example,in Tvrdik et al. 2000, JCB 149:707-717 or WO0244320.

No specific elongase has been described to date for the production ofDHA (C22:6 n-3) in organisms which do not naturally produce this fattyacid. Only elongases which provide C₂₀- or C₂₄-fatty acids have beendescribed to date. A Δ5-elongase activity has not been described todate.

Higher plants comprise polyunsaturated fatty acids such as linoleic acid(C18:2) and linolenic acid (C18:3). ARA, EPA and DHA are found not atall in the seed oil of higher plants, or only in miniscule amounts (E.Ucciani: Nouveau Dictionnaire des Huiles Végétales [New Dictionary ofVegetable Oils]. Technique & Documentation—Lavoisier, 1995. ISBN:2-7430-0009-0). However, the production of LCPUFAs in higher plants,preferably in oil crops such as oilseed rape, linseed, sunflower andsoybeans, would be advantageous since large amounts of high-qualityLCPUFAs for the food industry, animal nutrition and pharmaceuticalpurposes might be obtained economically in this way. To this end, it isadvantageous to introduce, into oil crops, genes which encode enzymes ofthe LCPUFA biosynthesis via recombinant methods and to express themtherein. These genes encode for example Δ6-desaturases, Δ6-elongases,Δ5-desaturases or Δ4-desaturases. These genes can advantageously beisolated from microorganisms and lower plants which produce LCPUFAs andincorporate them in the membranes or triacylglycerides. Thus, it hasalready been possible to isolate Δ6-desaturase genes from the mossPhyscomitrella patens and Δ6-elongase genes from P. patens and from thenematode C. elegans.

The first transgenic plants to comprise and express genes encodingLCPUFA biosynthesis enzymes and which produce LCPUFAs were described forthe first time, for example, in DE 102 19 203 (process for theproduction of polyunsaturated fatty acids in plants). However, theseplants produce LCPUFAs in amounts which require further optimization forprocessing the oils which are present in the plants.

To make possible the fortification of food and of feed with thesepolyunsaturated fatty acids, there is therefore a great need for meansand measures for a simple inexpensive production of thesepolyunsaturated fatty acids, specifically in eukaryotic systems. Theobject of the present invention would therefore be the provision of suchmeans and measures. This object is achieved by the use forms which aredescribed in the patent claims and hereinbelow.

The present invention thus relates to a polynucleotide comprising anucleic acid sequence selected from the group consisting of:

-   -   (a) nucleic acid sequence as shown in one of the SEQ ID NO. 1,        3, 5, 7, 9, 11, 13 or 15;    -   (b) nucleic acid sequence which codes for a polypeptide which        features an amino acid sequence as shown in one of SEQ ID NO. 2,        4, 6, 8, 10, 12, 14 or 16;    -   (c) nucleic acid sequence which codes for a polypeptide with at        least 70% identity to a polypeptide which is encoded by the        nucleic acid sequence of (a) or (b), where the polypeptide has        desaturase or elongase activity; and    -   (d) nucleic acid sequence for a fragment of a nucleic acid of        (a), (b) or (c), where the fragment codes for a polypeptide with        a desaturase or elongase activity.

According to the invention, the term “polynucleotide” relates topolynucleotides which comprise nucleic acid sequences which code forpolypeptides with desaturase or elongase activity. The desaturase orelongase activities are preferably required for the biosynthesis oflipids or fatty acids. Especially preferably, they take the form of thefollowing desaturase or elongase activities: Δ4-desaturase,Δ5-desaturase, Δ5-elongase, Δ6-desaturase, Δ6-elongase orΔ12-desaturase. The desaturases and/or elongases are preferably involvedin the synthesis of polyunsaturated fatty acids (PUFAs) and especiallypreferably in the synthesis of long-chain PUFAs (LCPUFAs).

Suitable detection systems for these desaturase or elongase activitiesare described in the examples or in WO2005/083053. Especiallypreferably, the above-mentioned activities are, as regards substratespecificities and conversion rates, those of the respective enzymes fromOstreococcus lucimarinus. The specific polynucleotides according to theinvention, i.e. the polynucleotides with a nucleic acid sequence asshown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15, were obtained fromOstreococcus lucimarinus.

Therefore, polynucleotides according to the invention are in particular:Polynucleotides which code for a polypeptide with Δ12-desaturaseactivity and which (i) comprise a nucleic acid sequence as shown in SEQID NO: 1 or 3, (ii) comprise a nucleic acid sequence which codes for apolypeptide as shown in SEQ ID NO: 2 or 4, (iii) comprise a nucleic acidsequence with at least 83% identity to one of the nucleic acid sequencesof (i) or (ii), or (iv) a nucleic acid sequence of a fragments of anucleic acid from (i), (ii) or (iii).

Polynucleotides which code for a polypeptide with Δ4-desaturase activityand which (i) comprise a nucleic acid sequence as shown in SEQ ID NO: 5,(ii) comprise a nucleic acid sequence which codes for a polypeptide asshown in SEQ ID NO: 6, (iii) comprise a nucleic acid sequence with atleast 72% identity to one of the nucleic acid sequences of (i) or (ii),or (iv) a nucleic acid sequence of a fragments of a nucleic acid from(i), (ii) or (iii).

Polynucleotides which code for a polypeptide with Δ5-desaturase activityand which (i) comprise a nucleic acid sequence as shown in SEQ ID NO: 7or 9, (ii) comprise a nucleic acid sequence which codes for apolypeptide as shown in SEQ ID NO: 8 or 10, (iii) comprise a nucleicacid sequence with at least 72% identity to one of the nucleic acidsequences of (i) or (ii), or (iv) a nucleic acid sequence of a fragmentof a nucleic acid from (i), (ii) or (iii).

Polynucleotides which code for a polypeptide with Δ5-elongase activityand which (i) comprise a nucleic acid sequence as shown in SEQ ID NO:11, (ii) comprise a nucleic acid sequence which codes for a polypeptideas shown in SEQ ID NO: 12, (iii) comprise a nucleic acid sequence withat least 78% identity to one of the nucleic acid sequences of (i) or(ii), or (iv) a nucleic acid sequence of a fragment of a nucleic acidfrom (i), (ii) or (iii).

Polynucleotides which code for a polypeptide with Δ6-desaturase activityand which (i) comprise a nucleic acid sequence as shown in SEQ ID NO:13, (ii) comprise a nucleic acid sequence which codes for a polypeptideas shown in SEQ ID NO: 14, (iii) comprise a nucleic acid sequence withat least 72% identity to one of the nucleic acid sequences of (i) or(ii), or (iv) a nucleic acid sequence of a fragment of a nucleic acidfrom (i), (ii) or (iii).

Polynucleotides which code for a polypeptide with Δ6-elongase activityand which (i) comprise a nucleic acid sequence as shown in SEQ ID NO:15, (ii) comprise a nucleic acid sequence which codes for a polypeptideas shown in SEQ ID NO: 16, (iii) comprise a nucleic acid sequence withat least 71% identity to one of the nucleic acid sequences of (i) or(ii), or (iv) a nucleic acid sequence of a fragment of a nucleic acidfrom (i), (ii) or (iii).

Naturally, the abovementioned specific sequences may, taking intoconsideration the degeneracy of the genetic code, also be modified,where the modified polynucleotides still code for polypeptides with anamino acid sequence as shown in any of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14or 16 which feature the abovementioned desaturase or elongaseactivities.

The term “polynucleotide” also comprises variants of the abovementionedspecific polynucleotides. These may be homologous, orthologous orparalogous sequences. Such variants comprise nucleic acid sequenceswhich feature at least one base substitution, one base addition or onebase deletion, it being intended that the variants still encode apolypeptide with the abovementioned biological activity of therespective starting sequence. Variants comprise polynucleotides whichare capable of hybridization, with the abovementioned polynucleotides,preferably under stringent conditions. Especially preferred stringentconditions are known to the skilled worker and can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N. Y. (1989),6.3.1-6.3.6. A preferred example of stringent hybridization conditionsare hybridizations in 6× sodium chloride/sodium citrate (=SSC) atapproximately 45° C., followed by one or more wash steps in 0.2×SSC,0.1% SDS at 50 to 65° C. The skilled worker knows that thesehybridization conditions differ regarding temperature and bufferconcentration, depending on the type of the nucleic acid and when forexample organic solvents are present. The temperature differs forexample under “standard hybridization conditions” as a function of thetype of the nucleic acid between 42° C. and 58° C. in an aqueous bufferat a concentration of from 0.1 to 5×SSC (pH 7.2). If organic solvent ispresent in the abovementioned buffer, for example 50% formamide, thetemperature under standard conditions is approximately 42° C.Preferably, the hybridization conditions for DNA:DNA hybrids are, forexample, 0.1×SSC and 20° C. to 45° C., preferably between 30° C. and 45°C. Preferably, the hybridization conditions for DNA:RNA hybrids are, forexample, 0.1×SSC and 30° C. to 55° C., preferably between 45° C. and 55°C. The abovementioned hybridization temperatures are determined forexample for a nucleic acid with a length of approximately 100 bp (=basepairs) and a G+C content of 50% in the absence of formamide. The skilledworker knows how the hybridization conditions required can be determinedby referring to textbooks such as the abovementioned textbooks, or fromthe following textbooks: Sambrook et al., “Molecular Cloning”, ColdSpring Harbor Laboratory, 1989; Hames and Higgins (Eds.) 1985, “NucleicAcids Hybridization: A Practical Approach”, IRL Press at OxfordUniversity Press, Oxford; Brown (Ed.) 1991, “Essential MolecularBiology: A Practical Approach”, IRL Press at Oxford University Press,Oxford. Alternatively, it is possible to provide variants of thespecific polynucleotides according to the invention by means ofprocesses which are based on the polymerase chain reaction (PCR). Tothis end, it is first possible to derive primers from conservedsequences (for example sequences which code for functional domains inthe polypeptide). Conserved sequences can be determined by sequencealignments with polynucleotides which code for polypeptides with asimilar activity. The template used may be DNA or cDNA from bacteria,fungi, plants or animals. DNA fragments which were obtained by PCR canbe used for screening suitable genomic libraries or cDNA libraries inorder—if required, to isolate, and to determine by sequencing, thecomplete open reading frame of the polynucleotide. Further variantscomprise polynucleotides which comprise a nucleic acid sequence with atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identity, or with any other percentageidentity mentioned herein, with one of the abovementioned specificnucleic acid sequences and which codes for a polypeptide with therespective biological activity. Equally comprised are polynucleotideswhich comprise nucleic acid sequences which code for a polypeptide withan amino acid sequence which has at least 70%, at least 75%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identity,or with any other percentage identity mentioned herein, with one of theabovementioned specific amino acid sequences and where the polypeptidehas the respective biological activity of the starting sequence. Thepercentage of identical nucleotides or amino acids preferably relates toa sequence segment of at least 50% of the sequences to be compared, andpreferably over the entire length of the sequences to be compared. Amultiplicity of programs which implement algorithms for such comparisonsare described in the prior art and commercially available. Inparticular, reference may be made to the algorithms of Needleman andWunsch or Smith and Waterman, which give particularly reliable results.These algorithms can preferably be implemented by the followingprograms: PileUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al.,CABIOS, 5 1989: 151-153), Gap and BestFit (Needleman and Wunsch (J. Mol.Biol. 48; 443-453 (1970)) and Smith and Waterman (Adv. Appl. Math. 2;482-489 (1981))), as part of the GCG software [Genetics Computer Group,575 Science Drive, Madison, Wis., USA 53711 (1991)]. For the purposes ofthe present invention, it is especially preferred to determine thepercentage (%) of the sequence identity with the GAP program over theentire sequence, with the following set parameters: Gap Weight: 50,Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000.

A polynucleotide which only comprises a fragment of the abovementionednucleic acid sequences is also a polynucleotide according to theinvention. Here, it is intended that the fragment codes for apolypeptide which features the biological activity of the startingsequence, or of the polypeptide which the latter codes for. Polypeptideswhich are encoded by such polynucleotides therefore comprise, or consistof, domains of the abovementioned specific polypeptides (startingpolypeptides) which confer the biological activity. A fragment for thepurposes of the invention preferably comprises at least 50, at least100, at least 250 or at least 500 consecutive nucleotides of theabovementioned specific sequences or codes for an amino acid sequencecomprising at least 20, at least 30, at least 50, at least 80, at least100 or at least 150 consecutive amino acids of one of the abovementionedspecific amino acid sequences.

The polynucleotide variants according to the invention preferablyfeature at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80% or at least 90% of therespective biological activity of the polypeptide which is encoded bythe starting sequence. That is to say the polypeptides which are encodedby the polynucleotides according to the invention can participate in themetabolism of compounds required for the synthesis of fatty acids, fattyacid esters such as diacylglycerides and/or triacylglycerides in anorganism, preferably in a plant or plant cell, or can participate in thetransport of molecules across membranes, which means C₁₈-, C₂₀- orC₂₂-carbon chains in the fatty acid molecule with double bonds at atleast two, advantageously three, four, five or six positions.

The polynucleotides according to the invention either comprise theabovementioned specific nucleic acid sequences or consist of them. Thisis to say that the polynucleotides according to the invention may, inprinciple, also comprise further nucleotides. These may preferably be3′- or 5′-untranslated regions of the genomic nucleic acid sequence.They preferably consist of at least 100, 200 or 500 nucleotides at the5′ terminus and of at least 20, 50 or 100 nucleotides at the 3′ terminusof the coding region. Further polynucleotides which comprise additionalnucleic acid sequences are those which code for fusion proteins. Suchfusion proteins can code for further polypeptide or polypeptideportions, in addition to the abovementioned polypeptides. The additionalpolypeptide or polypeptide portion may take the form of further enzymesof lipid or fatty acid biosynthesis. Others which are feasible arepolypeptides which may act as expression markers (green, yellow, red,blue fluorescent proteins, alkaline phosphatase and others) or so-called“tags” as labels or as an aid for purification (for example FLAG tags,6-histidine tags, MYC tags and others).

Polynucleotide variants can be isolated from different natural orartificial sources. For example, they can be generated artificially byin-vitro or in-vivo mutagenesis. Homologs or orthologs of the specificsequences can be obtained from a wide range of animals, plants ormicroorganisms. They are preferably obtained from algae. Especiallypreferred are algae of the family Prasinophyceae such as from the generaHeteromastix, Mammella, Mantoniella, Micromonas, Nephroselmis,Ostreococcus, Prasinocladus, Prasinococcus, Pseudoscourfielda,Pycnococcus, Pyramimonas, Scherffelia or Tetraselmis, such as of thegenera and species Heteromastix longifillis, Mamiella gilva, Mantoniellasquamata, Micromonas pusilla, Nephroselmis olivacea, Nephroselmispyriformis, Nephroselmis rotunda, Ostreococcus tauri, Ostreococcus sp.Prasinocladus ascus, Prasinocladus lubricus, Pycnococcus provasolii,Pyramimonas amylifera, Pyramimonas disomata, Pyramimonas obovata,Pyramimonas orientalis, Pyramimonas parkeae, Pyramimonas spinifera,Pyramimonas sp., Tetraselmis apiculata, Tetraselmis carteriaformis,Tetraselmis chui, Tetraselmis convolutae, Tetraselmis desikacharyi,Tetraselmis gracilis, Tetraselmis hazeni, Tetraselmis impellucida,Tetraselmis inconspicua, Tetraselmis levis, Tetraselmis maculata,Tetraselmis marina, Tetraselmis striata, Tetraselmis subcordiformis,Tetraselmis suecica, Tetraselmis tetrabrachia, Tetraselmis tetrathele,Tetraselmis verrucosa, Tetraselmis verrucosa fo. Rubens or Tetraselmissp. The polynucleotides are preferably derived from algae of the generaMantoniella and Ostreococcus. Equally preferred are algae such asIsochrysis or Crypthecodinium, algae/diatoms such as Thalassiosira,Phaeodactylum or Thraustochytrium, mosses such as Physcomitrella orCeratodon, very especially preferred are the algae of the generaMantoniella or Ostreococcus or the diatoms of the genera Thalassiosiraor Crypthecodinium. The polynucleotides can also be preferably obtainedfrom higher plants such as Primulaceae such as Aleuritia, Calendulastellata, Osteospermum spinescens or Osteospermum hyoseroides,microorganisms such as fungi, such as Aspergillus, Thraustochytrium,Phytophthora, Entomophthora, Mucor or Mortierella, bacteria such asShewanella, yeasts or animals such as nematodes, for examplecaenorhabditis, insects or fish. The polynucleotide variants are alsopreferably derived from an animal from the order vertebrates. Especiallypreferably, the polynucleotides are derived from the class Vertebrata;Euteleostomi, Actinopterygii; Neopterygii; Teleostei; Euteleostei,Protacanthopterygii, Salmoniformes; Salmonidae or Oncorhynchus and, veryespecially preferably, from the order Salmoniformes such as the familySalmonidae, such as the genus Salmo, for example from the genera andspecies Oncorhynchus mykiss, Trutta trutta or Salmo trutta fario. Here,the polynucleotides according to the invention can be isolated by meansof standard techniques of molecular biology and of the sequenceinformation provided herein. Also, it is possible, with the aid ofcomparative algorithms, to identify for example a homologous sequence orhomologous, conserved sequence regions at the DNA or amino acid level.These can be employed as hybridization probe and standard hybridizationtechniques (such as, for example, those described in Sambrook et al.,Molecular Cloning: A Laboratory Manual. 2^(nd) Ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989) for isolating further nucleic acid sequences which areuseful in the process. Moreover, it is possible to isolatepolynucleotides or fragments thereof by means of polymerase chainreaction (PCR), where oligonucleotide primers which are based on thissequence or parts thereof are employed (for example, a nucleic acidmolecule comprising the complete sequence or part thereof can beisolated by polymerase chain reaction using oligonucleotide primerswhich have been generated on the basis of the same sequence). Forexample, it is possible to isolate mRNA from cells (for example by theguanidinium thiocyanate extractive method by Chirgwin et al. (1979)Biochemistry 18:5294-5299) and cDNA can be generated by means of reversetranscriptase (for example Moloney MLV reverse transcriptase, obtainablefrom Gibco/BRL, Bethesda, Md., or AMV reverse transcriptase, obtainablefrom Seikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for the amplification by means of polymerasechain reaction can be generated on the basis of the amino acid sequencesshown in the SEQ ID numbers. A nucleic acid according to the inventioncan be amplified using cDNA or, alternatively, genomic DNA as thetemplate and suitable oligonucleotide primers, following standard PCRamplification techniques. The nucleic acid amplified thus can be clonedinto a suitable vector and characterized by means of DNA sequenceanalysis. Oligonucleotides which correspond to a desaturase nucleotidesequence can be generated by synthetic standard methods, for exampleusing an automatic DNA synthesizer.

The polynucleotides according to the invention can either be provided inthe form of isolated polynucleotides (i.e. isolated from their naturalorigin, for example the genomic locus) or else in genetically modifiedform (i.e. the polynucleotides may also be present at their naturalgenetic locus, but, in such a case, must be genetically modified). Anisolated polynucleotide preferably comprises less than 5 kb, 4 kb, 3 kb,2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleic acid sequence which occursnaturally in its environment. The polynucleotide according to theinvention may be present as a single-stranded or double-stranded nucleicacid molecule and may take the form of genomic DNA, cDNA or RNA. Thepolynucleotides according to the invention comprise all orientations ofthe sequences shown in the SEQ ID numbers, i.e. also complementarystrands and reverse, or reverse-complementary, orientations. The termfurthermore also comprises chemically modified nucleic acids, such asthe naturally occurring methylated DNA molecules, or artificial nucleicacids, for example biotinylated nucleic acids.

The invention also comprises oligonucleotides of at least 15 bp,preferably at least 20 bp, at least 25 bp, at least 30 bp, at least 35bp or at least 50 bp, which are capable of specifically hybridizingunder stringent conditions with one of the abovementionedpolynucleotides. The oligonucleotides may consist of DNA or RNA or both.Such oligonucleotides can be employed as primers for the PCR, asexpression-inhibitory antisense oligonucleotides, for RNA interference(RNAi) approaches or for chimeroplastic or genoplastic approaches. RNAimethods are described for example in Fire et al., Nature (1998)391:806-811; Fire, Trends Genet. 15, 358-363 (1999); Sharp, RNAinterference 2001. Genes Dev. 15,485-490 (2001); Hammond et al. NatureRev. Genet. 2, 1110-1119 (2001); Tuschl, Chem. Biochem. 2, 239-245(2001); Hamilton et al., Science 286, 950-952 (1999); Hammond et al.,Nature 404, 293-296 (2000); Zamore et al., Cell 101, 25-33 (2000);Bernstein et al., Nature 409, 363-366 (2001); Elbashir et al., GenesDev. 15, 188-200 (2001); WO 01/29058; WO 99/32619; or Elbashir et al.,2001 Nature 411: 494-498 and serve for inhibiting gene expression bydegrading the mRNA. Chimeroplastic or genoplastic approaches serve thein-vivo modification (for example the introduction of point mutations)into genes at their endogenous loci. Such methods are disclosed in U.S.Pat. No. 5,565,350, U.S. Pat. No. 5,756,325, U.S. Pat. No. 5,871,984,U.S. Pat. No. 5,731,181, U.S. Pat. No. 5,795,972, U.S. Pat. No.6,573,046, U.S. Pat. No. 6,211,351, U.S. Pat. No. 6,586,184, U.S. Pat.No. 6,271,360 and U.S. Pat. No. 6,479,292.

Advantageously, it has emerged that the polynucleotides according to theinvention can be employed particularly efficiently for the recombinantproduction of polyunsaturated fatty acids in host cells and transgenicorganisms. In particular, the polypeptides encoded by thepolynucleotides according to the invention, which have Δ12-desaturase,Δ4-desaturase, Δ5-desaturase, Δ5-elongase, Δ6-desaturase and Δ6-elongaseactivity, are capable of converting C₁₈-, C₂₀- and C₂₂-fatty acids withone, two, three, four or five double bonds, and preferablypolyunsaturated C₁₈-fatty acids with one, two or three double bonds suchas C18:1^(Δ9), C18:2^(Δ9,12) or C18:3^(Δ9,12,15) polyunsaturatedC₂₀-fatty acids with three or four double bonds such as C20:3^(Δ8,11,14)or C20:4^(Δ8,11,14,17) or polyunsaturated C₂₂-fatty acids with four orfive double bonds such as C22:4^(Δ7,10,13,16) or C22:5^(Δ7,10,13,16,19).Preferably, it is the fatty acids in phospholipids or CoA fatty acidesters which are desaturated, advantageously in the CoA fatty acidesters. Thus, a simple, inexpensive production of these polyunsaturatedfatty acids is possible, specifically in eukaryotic systems. Theunsaturated fatty acids produced by means of the polynucleotidesaccording to the invention can then be formulated as oil, lipid andfatty acid compositions and can be employed in a suitable manner.

The present invention furthermore relates to a vector which comprisesthe polynucleotide according to the invention.

The term “vector” refers to a nucleic acid molecule which is capable oftransporting another nucleic acid molecule, such as the polynucleotidesaccording to the invention, to which it is bound. One type of vector isa “plasmid”, a circular double-stranded DNA loop into which additionalDNA segments can be ligated. A further type of vector is a viral vector,it being possible for additional DNA segments to be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they have been introduced (for example bacterialvectors with bacterial replication origin). Other vectors areadvantageously integrated into the genome of a host cell when they areintroduced into the host cell, and thus replicate together with the hostgenome. Moreover, certain vectors can govern the expression of geneswith which they are in operable linkage. These vectors are referred toin the present context as “expression vectors”. Usually, expressionvectors which are suitable for DNA recombination techniques take theform of plasmids. In the present description, “plasmid” and “vector” canbe used exchangeably since the plasmid is the form of vector which ismost frequently used. However, the invention is also intended tocomprise other forms of expression vectors, such as viral vectors, whichexert similar functions. Furthermore, the term “vector” is also intendedto comprise other vectors with which the skilled worker is familiar,such as phages, viruses such as SV40, CMV, TMV, transposons, ISelements, phasmids, phagemids, cosmids, linear or circular DNA,artificial chromosomes. Finally, the term also comprises constructs forthe targeted, i.e. homologous, recombination, or the heterologousinsertion of polynucleotides.

Vectors can be introduced into prokaryotic and eukaryotic cells viaconventional transformation or transfection techniques. The terms“transformation” and “transfection”, conjugation and transduction, asused in the present context, are intended to comprise a multiplicity ofmethods known in the prior art for the introduction of foreign nucleicacid (for example DNA) into a host cell, including calcium phosphate orcalcium chloride coprecipitation, DEAE-dextran-mediated transfection,lipofection, natural competence, chemically mediated transfer,electroporation or particle bombardment. Suitable methods for thetransformation or transfection of host cells, including plant cells, canbe found in Sambrook et al. (Molecular Cloning: A Laboratory Manual.,2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989) and other laboratory textbookssuch as Methods in Molecular Biology, 1995, Vol. 44, Agrobacteriumprotocols, Ed.: Gartland and Davey, Humana Press, Totowa, N.J.

Suitable cloning vectors are generally known to the skilled worker. Inparticular, they include vectors which can replicate in microbialsystems, that is mainly vectors which ensure efficient cloning in yeastsor fungi and which make possible the stable transformation of plants.Those which must be mentioned are in particular various binary andcointegrated vector systems which are suitable for the T-DNA-mediatedtransformation. Such vector systems are, as a rule, characterized inthat they comprise at least the vir genes, which are required for theagrobacterium-mediated transformation, and the T-DNA-bordering sequences(T-DNA border). Preferably, these vector systems also comprise furthercis-regulatory regions such as promoters and terminators and/orselection markers, by means of which suitably transformed organisms canbe identified. While in the case of cointegrated vector systems virgenes and T-DNA sequences are arranged on the same vector, binarysystems are based on at least two vectors, one of which bears vir genes,but no T-DNA, and the second vector bears T-DNA, but no vir genes. As aresult, the last-mentioned vectors are relatively small, easy tomanipulate and to replicate both in E. coli and in Agrobacterium. Thesebinary vectors include vectors from the pBIB-HYG series, the pPZPseries, the pBecks series and the pGreen series. Preferably usedaccording to the invention are Bin19, pBl101, pBinAR, pGPTV and pCAMBIA.An overview over binary vectors and their use is found in Hellens et al,Trends in Plant Science (2000) 5, 446-451. The vectors with the insertedpolynucleotides according to the invention can be propagated stablyunder selective conditions in microorganisms, in particular Escherichiacoli and Agrobacterium tumefaciens, and make possible a transfer ofheterologous DNA into plants or microorganisms. The polynucleotidesaccording to the invention can be introduced into organisms such asmicroorganisms or plants by means of the cloning vectors and thus usedfor transforming plants. Vectors which are suitable for this purpose arepublished in: Plant Molecular Biology and Biotechnology (CRC Press, BocaRaton, Fla.), chapter 6/7, p. 71-119 (1993); F. F. White, Vectors forGene Transfer in Higher Plants; in: Transgenic Plants, vol. 1,Engineering and Utilization, eds.: Kung and R. Wu, Academic Press, 1993,15-38; B. Jenes et al., Techniques for Gene Transfer, in: TransgenicPlants, vol. 1, Engineering and Utilization, eds.: Kung and R. Wu,Academic Press (1993), 128-143; Potrykus, Annu. Rev. Plant Physiol.Plant Molec. Biol. 42 (1991), 205-225)).

The vector is preferably an expression vector. The polynucleotide ispresent in the expression vector according to the invention in operative(i.e. functional) linkage with an expression control sequence. Theexpression control sequence together with the polynucleotide andoptionally further sequence elements of the vector is also referred toas the expression cassette. The expression control sequence ensuresthat, after transformation or transfection into a host cell, thepolynucleotide can be expressed.

The expression control sequence to be used preferably comprisescis-regulatory elements such as promoter and/or enhancer nucleic acidsequences, which are recognized by the transcription machinery of thehost cells. The term furthermore comprises other expression controlelements, for example polyadenylation signals and RNA-stabilizingsequences. These regulatory sequences are described for example inGoeddel: Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) or see: Gruber and Crosby, in: Methodsin Plant Molecular Biology and Biotechnolgy, CRC Press, Boca Raton,Fla., eds.: Glick and Thompson, chapter 7, 89-108, including theliterature cited therein. Expression control sequences comprise thosewhich govern the constitutive expression of a nucleotide sequence inmany types of host cells, and those which govern the direct expressionof the nucleotide sequence only in certain host cells under certainconditions. The skilled worker knows that the design of the expressionvector may depend on factors such as the choice of the host cell to betransformed, the extent of the expression of the desired protein and thelike. The polynucleotides according to the invention may be present inone or more copies in the expression cassette or in the expressionvector according to the invention (for example in the form of severalexpression cassettes). Here, the regulatory sequences or factors canhave a positive effect on, preferably the gene expression of theintroduced genes, as described above, and thereby increase it. Thus, itis possible to enhance the regulatory elements advantageously at thetranscription level by using strong transcription signals such aspromoters and/or “enhancers”.

Besides, it is also possible to enhance the translation, for example byimproving the mRNA stability. Further expression control sequenceswithin the meaning of the present invention are translation terminatorsat the 3′ end of the polynucleotides to be translated. An example of aterminator which can be used here is the OCS1 terminator. As in the caseof the promoters, a different terminator sequence should be used foreach of the polynucleotides to be expressed.

Preferred expression control sequences or regulatory sequences arepresent in promoters such as the cos, tac, trp, tet, trp-tet, Ipp, lac,Ipp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6, λ-PR or λ-PL promotersand are advantageously employed in Gram-negative bacteria. Furtheradvantageous regulatory sequences are, for example, present in theGram-positive promoters amy and SPO2, in the yeast or fungal promotersADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plantpromoters CaMV/35S [Franck et al., Cell 21 (1980) 285-294], PRP1 [Wardet al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, lib4, usp, STLS1, B33,nos or in the ubiquitin or phaseolin promoter. Advantageous in thiscontext are also inducible promoters, such as the promoters described inEP-A-0 388 186 (benzenesulfonamide-inducible), Plant J. 2, 1992:397-404(Gatz et al., tetracycline-inducible), EP-A-0 335 528 (abscissicacid-inducible) or WO 93/21334 (ethanol- or cyclohexenol-inducible)promoters.

Further suitable plant promoters are the cytosolic FBPase promoter orthe ST-LSI promoter of potato (Stockhaus et al., EMBO J. 8, 1989, 2445),the glycine max phosphoribosylpyrophosphate amidotransferase promoter(Genbank Accession No. U87999) or the node-specific promoter describedin EP-A-0 249 676. Especially advantageous promoters are promoters whichmake possible the expression in tissues which are involved in thebiosynthesis of fatty acids. Very especially advantageous areseed-specific promoters, such as the USP promoter as described, but alsoother promoters such as the LeB4, DC3, phaseolin or napin promoter.Further especially advantageous promoters are seed-specific promoterswhich can be used for monocotyledonous or dicotyledonous plants andwhich are described in U.S. Pat. No. 5,608,152 (oilseed rape napinpromoter), WO 98/45461 (Arobidopsis oleosin promoter), U.S. Pat. No.5,504,200 (Phaseolus vulgaris phaseolin promoter), WO 91/13980 (BrassicaBce4 promoter), by Baeumlein et al., Plant J., 2, 2, 1992:233-239 (LeB4promoter from a legume), these promoters being suitable for dicots.Examples of promoters which are suitable for monocots are the barleyIpt-2 or Ipt-1 promoter (WO 95/15389 and WO 95/23230), the barleyhordein promoter and other suitable promoters described in WO 99/16890.In principle, it is possible to use all natural promoters together withtheir regulatory sequences, such as those mentioned above, as expressioncontrol sequences. It is also possible to use synthetic promoters,either in addition or alone, in particular when they mediateseed-specific expression, such as those described in WO 99/16890.

In order to achieve a particularly high PUFA content, especially intransgenic plants, the polynucleotides of the present invention shouldpreferably be expressed in oil crops in a seed-specific manner. To thisend, seed-specific promoters can be used, or those promoters which areactive in the embryo and/or in the endosperm. In principle,seed-specific promoters can be isolated both from dicotyledonous andfrom monocotyledonous plants. Advantageous preferred promoters arelisted hereinbelow: USP (=unknown seed protein) and vicilin (Vicia faba)[Baumlein et al., Mol. Gen Genet., 1991, 225(3)], napin (oilseed rape)[U.S. Pat. No. 5,608,152], acyl carrier protein (oilseed rape) [U.S.Pat. No. 5,315,001 and WO 92/18634], oleosin (Arabidopsis thaliana) [WO98/45461 and WO 93/20216], phaseolin (Phaseolus vulgaris) [U.S. Pat. No.5,504,200], Bce4 [WO 91/13980], legumines B4 (LegB4 promoter) [Baumleinet al., Plant J., 2,2, 1992], Lpt2 and Ipt1 (barley) [WO 95/15389 andWO95/23230], seed-specific promoters from rice, maize and wheat [WO99/16890], Amy32b, Amy 6-6 and aleurain [U.S. Pat. No. 5,677,474], Bce4(oilseed rape) [U.S. Pat. No. 5,530,149], glycinin (soybean) [EP 571741], phosphoenol pyruvate carboxylase (soybean) [JP 06/62870], ADR12-2(soybean) [WO 98/08962], isocitrate lyase (oilseed rape) [U.S. Pat. No.5,689,040] or α-amylase (barley) [EP 781 849].

Plant gene expression can also be facilitated via a chemically induciblepromoter (see review in Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol.Biol., 48:89-108). Chemically inducible promoters are particularlysuitable when it is desired that gene expression should take place in atime-specific manner. Examples of such promoters are a salicylicacid-inducible promoter (WO 95/19443), a tetracycline-inducible promoter(Gatz et al. (1992) Plant J. 2, 397-404) and an ethanol-induciblepromoter.

To ensure the stable integration of the various biosynthesis genes intothe transgenic plant over a plurality of generation, each of thepolynucleotides according to the invention should be expressed under thecontrol of a separate promoter, preferably a promoter which differs fromthe other promoters, since repeating sequence motifs can lead toinstability of the T-DNA, or to recombination events. In this context,the expression cassette is advantageously constructed in such a way thata promoter is followed by a suitable cleavage site, advantageously in apolylinker, for insertion of the nucleic acid to be expressed and, ifappropriate, a terminator is then positioned behind the polylinker. Thissequence is repeated several times, preferably three, four or fivetimes, so that up to five genes can be combined in one construct andintroduced into the transgenic plant in order to be expressed.Advantageously, the sequence is repeated up to three times. To expressthe nucleic acid sequences, the latter are inserted behind the promotervia a suitable cleavage site, for example in the polylinker.

Advantageously, each nucleic acid sequence has its own promoter and, ifappropriate, its own terminator. Such advantageous constructs aredisclosed, for example, in DE 101 02 337 or DE 101 02 338. However, itis also possible to insert a plurality of nucleic acid sequences behinda promoter and, if appropriate, before a terminator. Here, the insertionsite, or the sequence, of the inserted nucleic acids in the expressioncassette is not of critical importance, that is to say a nucleic acidsequence can be inserted at the first or last position in the cassettewithout its expression being substantially influenced thereby.Advantageously, different promoters such as, for example, the USP, LegB4or DC3 promoter, and different terminators can be used in the expressioncassette. However, it is also possible to use only one type of promoterin the cassette. This, however, may lead to undesired recombinationevents.

The recombinant expression vectors used can be designed for theexpression in prokaryotic or eukaryotic cells. This is advantageoussince intermediate steps of the vector construction are frequentlycarried out in microorganisms for the sake of simplicity. For example,the Δ12-desaturase, Δ6-desaturase, Δ6-elongase, Δ5-desaturase,Δ5-elongase and/or Δ4-desaturase genes can be expressed in bacterialcells, insect cells (using Baculovirus expression vectors), yeast andother fungal cells (see Romanos, M. A., et al. (1992) “Foreign geneexpression in yeast: a review”, Yeast 8:423-488; van den Hondel, C. A.M. J. J., et al. (1991) “Heterologous gene expression in filamentousfungi”, in: More Gene Manipulations in Fungi, J. W. Bennet & L. L.Lasure, Ed., pp. 396-428: Academic Press: San Diego; and van den Hondel,C. A. M. J. J., & Punt, P. J. (1991) “Gene transfer systems and vectordevelopment for filamentous fungi, in: Applied Molecular Genetics ofFungi, Peberdy, J. F., et al., Ed., pp. 1-28, Cambridge UniversityPress: Cambridge), algae (Falciatore et al., 1999, Marine Biotechnology.1, 3:239-251), ciliates of the types: Holotrichia, Peritrichia,Spirotrichia, Suctoria, Tetrahymena, Paramecium, Colpidium, Glaucoma,Platyophrya, Potomacus, Desaturaseudocohnilembus, Euplotes,Engelmaniella and Stylonychia, in particular of the genus Stylonychialemnae, using vectors in a transformation method as described in WO98/01572 and, preferably, in cells of multi-celled plants (see Schmidt,R. and Willmitzer, L. (1988) “High efficiency Agrobacteriumtumefaciens-mediated transformation of Arabidopsis thaliana leaf andcotyledon explants” Plant Cell Rep.:583-586; Plant Molecular Biology andBiotechnology, C Press, Boca Raton, Fla., Chapter 6/7, pp. 71-119(1993); F. F. White, B. Jenes et al., Techniques for Gene Transfer, in:Transgenic Plants, Vol. 1, Engineering and Utilization, Ed.: Kung and R.Wu, Academic Press (1993), 128-43; Potrykus, Annu. Rev. Plant Physiol.Plant Molec. Biol. 42 (1991), 205-225 (and references cited therein)).Suitable host cells are furthermore discussed in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). As an alternative, the recombinant expressionvector can be transcribed and translated in vitro, for example usingT7-promoter regulatory sequences and T7-polymerase.

In most cases, the expression of proteins in prokaryotes involves theuse of vectors comprising constitutive or inducible promoters whichgovern the expression of fusion or nonfusion proteins. Typical fusionexpression vectors are, inter alia, pGEX (Pharmacia Biotech Inc; Smith,D. B., and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) und pRIT5 (Pharmacia, Piscataway, N.J.), whereglutathione S-transferase (GST), maltose-E-binding protein and proteinA, respectively, is fused with the recombinant target protein. Examplesof suitable inducible nonfusion E. coli expression vectors are, interalia, pTrc (Amann et al. (1988) Gene 69:301-315) and pET 11d (Studier etal., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). The target gene expression fromthe pTrc vector is based on the transcription from a hybrid trp-lacfusion promoter by the host RNA polymerase. The target gene expressionfrom the vector pET 11d is based on the transcription of a T7-gn10-lacfusion promoter, which is mediated by a viral RNA polymerase (T7 gn1),which is coexpressed. This viral polymerase is provided by the hoststrains BL21 (DE3) or HMS174 (DE3) from a resident λ-prophage whichharbors a T7 gn1 gene under the transcriptional control of the lacUV 5promoter.

Other vectors which are suitable for prokaryotic organisms are known tothe skilled worker, these vectors are, for example in E. coli pLG338,pACYC184, the pBR series such as pBR322, the pUC series such as pUC18 orpUC19, the M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24,pLG200, pUR290, plN-111113-B1, λgt11 or pBdCI, in Streptomyces pIJ101,pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214, inCorynebacterium pSA77 or pAJ667.

In a further embodiment, the expression vector is a yeast expressionvector. Examples for vectors for expression in the yeast S. cerevisiaecomprise pYeDesaturasecl (Baldari et al. (1987) Embo J. 6:229-234), pMFa(Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al.(1987) Gene 54:113-123) and pYES2 (Invitrogen Corporation, San Diego,Calif.). Vectors and processes for the construction of vectors which aresuitable for use in other fungi, such as the filamentous fungi, comprisethose which are described in detail in: van den Hondel, C. A. M. J. J.,& Punt, P. J. (1991) “Gene transfer systems and vector development forfilamentous fungi, in: Applied Molecular Genetics of fungi, J. F.Peberdy et al., Ed., pp. 1-28, Cambridge University Press: Cambridge, orin: More Gene Manipulations in Fungi [J. W. Bennet & L. L. Lasure, Ed.,pp. 396-428: Academic Press: San Diego]. Further suitable yeast vectorsare, for example, pAG-1, YEp6, YEp13 or pEMBLYe23.

As an alternative, the polynucleotides according to the invention canalso be expressed in insect cells using Baculovirus expression vectors.Baculovirus vectors which are available for the expression of proteinsin cultured insect cells (for example Sf9 cells) comprise the pAc series(Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series(Lucklow and Summers (1989) Virology 170:31-39).

Preferred plant expression vectors comprise those which are described indetail in: Becker, D., Kemper, E., Schell, J., and Masterson, R. (1992)“New plant binary vectors with selectable markers located proximal tothe left border”, Plant Mol. Biol. 20:1195-1197; and Bevan, M. W. (1984)“Binary Agrobacterium vectors for plant transformation”, Nucl. AcidsRes. 12:8711-8721; Vectors for Gene Transfer in Higher Plants; in:Transgenic Plants, Vol. 1, Engineering and Utilization, Ed.: Kung and R.Wu, Academic Press, 1993, p. 15-38. A plant expression cassettepreferably comprises expression control sequences which are capable ofgoverning the expression of genes in plant cells and which are linkedoperably so that each sequence can fulfill its function, such astranscriptional termination, for example polyadenylation signals.Preferred polyadenylation signals are those which are derived fromAgrobacterium tumefaciens T-DNA, such as gene 3 of the Ti plasmidpTiACH5 (Gielen et al., EMBO J. 3 (1984) 835 et seq.), which is known asoctopine synthase, or functional equivalents thereof, but all otherterminators which are functionally active in plants are also suitable.Since plant gene expression is very often not limited to thetranscriptional level, a plant expression cassette preferably comprisesother sequences which are linked operably, such as translationenhancers, for example the overdrive sequence, which comprises thetobacco mosaic virus 5′-untranslated leader sequence, which increasesthe protein/RNA ratio (Gallie et al., 1987, Nucl. Acids Research15:8693-8711). As described above, plant gene expression must be linkedoperably with a suitable promoter which triggers gene expression withthe correct timing or in a cell- or tissue-specific manner. Utilizablepromoters are constitutive promoters (Benfey et al., EMBO J. 8 (1989)2195-2202), such as those which are derived from plant viruses, such as35S CAMV (Franck et al., Cell 21 (1980) 285-294), 19S CaMV (see alsoU.S. Pat. No. 5,352,605 and WO 84/02913), or plant promoters, such asthe promoter of the small Rubisco subunit, which is described in U.S.Pat. No. 4,962,028. Other preferred sequences for use in operablelinkage in plant gene expression cassettes are targeting sequences,which are required for steering the gene product into its correspondingcell compartment (see a review in Kermode, Crit. Rev. Plant Sci. 15, 4(1996) 285-423 and references cited therein), for example into thevacuole, into the nucleus, all types of plastids, such as amyloplasts,chloroplasts, chromoplasts, the extracellular space, the mitochondria,the endoplasmid reticulum, oil bodies, peroxisomes and othercompartments of plant cells.

As described above, plant gene expression can also be achieved via achemically inducible promoter (see review in Gatz 1997, Annu. Rev. PlantPhysiol. Plant Mol. Biol., 48:89-108). Chemically inducible promotersare particularly suitable when it is desired that the gene expressiontakes place in a time-specific manner. Examples of such promoters are asalicylic-acid-inducible promoter (WO 95/19443), a tetracyclin-induciblepromoter (Gatz et al. (1992) Plant J. 2, 397-404) and anethanol-inducible promoter. Promoters which respond to biotic or abioticstress conditions are also suitable, for example the pathogen-inducedPRP1 gene promoter (Ward et al., Plant. Mol. Biol. 22 (1993) 361-366),the heat-inducible tomato hsp80 promoter (U.S. Pat. No. 5,187,267), thechill-inducible potato alpha-amylase promoter (WO 96/12814) or thewound-inducible pinll promoter (EP-A-0 375 091).

Especially preferred are those promoters which bring about the geneexpression in tissues and organs in which the biosynthesis of fattyacids, lipids and oils takes place, in seed cells, such as cells of theendosperm and of the developing embryo. Suitable promoters are theoilseed rape napin gene promoter (U.S. Pat. No. 5,608,152), the Viciafaba USP promoter (Baeumlein et al., Mol Gen Genet, 1991, 225(3):459-67), the Arabidopsis oleosin promoter (WO 98/45461), thePhaseolus vulgaris phaseolin promoter (U.S. Pat. No. 5,504,200), theBrassica Bce4 promoter (WO 91/13980) or the legumine B4 promoter (LeB4;Baeumlein et al., 1992, Plant Journal, 2 (2):233-9), and promoters whichbring about the seed-specific expression in monocotyledonous plants suchas maize, barley, wheat, rye, rice and the like. Suitable noteworthypromoters are the barley Ipt2 or Ipt1 gene promoter (WO 95/15389 and WO95/23230) or the promoters from the barley hordein gene, the riceglutelin gene, the rice oryzin gene, the rice prolamine gene, the wheatgliadine gene, the wheat glutelin gene, the maize zeine gene, the oatglutelin gene, the sorghum kasirin gene or the rye secalin gene, whichare described in WO 99/16890. Also especially suitable promoters arethose which lead to the plastid-specific expression, since plastids arethe compartment in which the precursors and some of the end products oflipid biosynthesis are synthesized. Suitable promoters, such as theviral RNA polymerase promoter, are described in WO 95/16783 and WO97/06250, and the clpP promotor from Arabidopsis, described in WO99/46394.

The abovementioned vectors are only a small overview over possiblevectors which are suitable. Further plasmids are known to the skilledworker and are described for example in: Cloning Vectors (eds. Pouwels,P. H., et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444904018). Further suitable expression systems for prokaryotic andeukaryotic cells, see chapters 16 and 17 of Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2^(nd)ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989.

As described above, the expression vector can also comprise furthergenes which are to be introduced into the organisms. It is possible andpreferred to introduce into the host organisms, and express in them,regulatory genes, such as genes for inductors, repressors or enzymeswhich, as a result of their enzymatic activity, engage in the regulationof one or more genes of a biosynthetic pathway. These genes can be ofheterologous or homologous origin. Heterologous genes or polynucleotidesare derived from a starting organism which differs from the targetorganism into which the genes or polynucleotides are to be introduced.In the case of homologous genes or polynucleotides, target organism andstarting organism are identical. The vector therefore preferablycomprises at least one further polynucleotide which codes for a furtherenzyme which is involved in the biosynthesis of lipids or fatty acids.The enzyme is preferably selected from the group consisting of: acyl-CoAdehydrogenase(s), acyl-ACP [=acyl carrier protein]desaturase(s),acyl-ACP thioesterase(s), fatty acid acyltransferase(s),acyl-CoA:lysophospholipid acyltransferase(s), fatty acid synthase(s),fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s),acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acidacetylenase(s), lipoxygenase(s), triacylglycerol lipase(s), allene oxidesynthase(s), hydroperoxide lyase(s), fatty acid elongase(s),Δ4-desaturase(s), Δ5-desaturase(s), Δ6-desaturase(s), Δ8-desaturase(s),Δ9-desaturase(s), Δ12-desaturase(s), Δ5-elongase(s), Δ6-elongase(s) andΔ9-elongase(s).

The invention also relates to a host cell which comprises thepolynucleotide according to the invention or the vector according to theinvention.

In principle, host cells for the purposes of the present invention maybe all eukaryotic or prokaryotic cells. They may be primary cells fromanimals, plants or multi-celled microorganisms, for example from thosewhich are mentioned in another place in the description. The termfurthermore also comprises cell lines which can be obtained from theseorganisms.

However, host cells for the purposes of the invention may also besingle-celled microorganisms, for example bacteria or fungi. Especiallypreferred microorganisms are fungi selected from the group of thefamilies Chaetomiaceae, Choanephoraceae, Cryptococcaceae,Cunninghamellaceae, Demetiaceae, Moniliaceae, Mortierellaceae,Mucoraceae, Pythiaceae, Sacharomycetaceae, Saprolegniaceae,Schizosacharomycetaceae, Sodariaceae or Tuberculariaceae. Furtherpreferred microorganisms are selected from the group: Choanephoraceae,such as the genera Blakeslea, Choanephora, for example the genera andspecies Blakeslea trispora, Choanephora cucurbitarum, Choanephorainfundibulifera var. cucurbitarum, Mortierellaceae, such as the genusMortierella, for example the genera and species Mortierella isabellina,Mortierella polycephala, Mortierella ramanniana, Mortierella vinacea,Mortierella zonata, Pythiaceae, such as the genera Phytium,Phytophthora, for example the genera and species Pythium debaryanum,Pythium intermedium, Pythium irregulare, Pythium megalacanthum, Pythiumparoecandrum, Pythium sylvaticum, Pythium ultimum, Phytophthoracactorum, Phytophthora cinnamomi, Phytophthora citricola, Phytophthoracitrophthora, Phytophthora cryptogea, Phytophthora drechsleri,Phytophthora erythroseptica, Phytophthora lateralis, Phytophthoramegasperma, Phytophthora nicotianae, Phytophthora nicotianae var.parasitica, Phytophthora palmivora, Phytophthora parasitica,Phytophthora syringae, Saccharomycetaceae, such as the genera Hansenula,Pichia, Saccharomyces, Saccharomycodes, Yarrowia, for example the generaand species Hansenula anomala, Hansenula californica, Hansenulacanadensis, Hansenula capsulata, Hansenula ciferrii, Hansenulaglucozyma, Hansenula henricii, Hansenula holstii, Hansenula minuta,Hansenula nonfermentans, Hansenula philodendri, Hansenula polymorpha,Hansenula saturnus, Hansenula subpelliculosa, Hansenula wickerhamii,Hansenula wingei, Pichia alcoholophila, Pichia angusta, Pichia anomala,Pichia bispora, Pichia burtonii, Pichia canadensis, Pichia capsulata,Pichia carsonii, Pichia cellobiosa, Pichia ciferrii, Pichia farinosa,Pichia fermentans, Pichia finlandica, Pichia glucozyma, Pichiaguilliermondii, Pichia haplophila, Pichia henricii, Pichia holstii,Pichia jadinii, Pichia lindnerii, Pichia membranaefaciens, Pichiamethanolica, Pichia minuta var. minuta, Pichia minuta var.nonfermentans, Pichia norvegensis, Pichia ohmeri, Pichia pastoris,Pichia philodendri, Pichia pini, Pichia polymorpha, Pichia quercuum,Pichia rhodanensis, Pichia sargentensis, Pichia stipitis, Pichiastrasburgensis, Pichia subpelliculosa, Pichia toletana, Pichiatrehalophila, Pichia vini, Pichia xylosa, Saccharomyces aceti,Saccharomyces bailii, Saccharomyces bayanus, Saccharomyces bisporus,Saccharomyces capensis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces cerevisiae var. ellipsoideus, Saccharomyceschevalieri, Saccharomyces delbrueckii, Saccharomyces diastaticus,Saccharomyces drosophilarum, Saccharomyces elegans, Saccharomycesellipsoideus, Saccharomyces fermentati, Saccharomyces florentinus,Saccharomyces fragilis, Saccharomyces heterogenicus, Saccharomyceshienipiensis, Saccharomyces inusitatus, Saccharomyces italicus,Saccharomyces kluyveri, Saccharomyces krusei, Saccharomyces lactis,Saccharomyces marxianus, Saccharomyces microellipsoides, Saccharomycesmontanus, Saccharomyces norbensis, Saccharomyces oleaceus, Saccharomycesparadoxus, Saccharomyces pastorianus, Saccharomyces pretoriensis,Saccharomyces rosei, Saccharomyces rouxii, Saccharomyces uvarum,Saccharomycodes ludwigii, Yarrowia lipolytica, Schizosaccharomycetaceaesuch as the genera Schizosaccharomyces e.g. the speciesSchizosaccharomyces japonicus var. japonicus, Schizosaccharomycesjaponicus var. versatilis, Schizosaccharomyces malidevorans,Schizosaccharomyces octosporus, Schizosaccharomyces pombe var.malidevorans, Schizosaccharomyces pombe var. pombe, Thraustochytriaceaesuch as the genera Althornia, Aplanochytrium, Japonochytrium,Schizochytrium, Thraustochytrium e.g. the species Schizochytriumaggregatum, Schizochytrium limacinum, Schizochytrium mangrovei,Schizochytrium minutum, Schizochytrium octosporum, Thraustochytriumaggregatum, Thraustochytrium amoeboideum, Thraustochytrium antacticum,Thraustochytrium arudimentale, Thraustochytrium aureum, Thraustochytriumbenthicola, Thraustochytrium globosum, Thraustochytrium indicum,Thraustochytrium kerguelense, Thraustochytrium kinnei, Thraustochytriummotivum, Thraustochytrium multirudimentale, Thraustochytriumpachydermum, Thraustochytrium proliferum, Thraustochytrium roseum,Thraustochytrium rossii, Thraustochytrium striatum or Thraustochytriumvisurgense.

Equally preferred as microorganisms are bacteria selected from the groupof the families Bacillaceae, Enterobacteriacae or Rhizobiaceae. It isespecially preferred to mention the following bacteria selected from thegroup: Bacillaceae, such as the genus Bacillus, for example the generaand species Bacillus acidocaldarius, Bacillus acidoterrestris, Bacillusalcalophilus, Bacillus amyloliquefaciens, Bacillus amylolyticus,Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacilluscoagulans, Bacillus sphaericus subsp. fusiformis, Bacillusgalactophilus, Bacillus globisporus, Bacillus globisporus subsp.marinus, Bacillus halophilus, Bacillus lentimorbus, Bacillus lentus,Bacillus licheniformis, Bacillus megaterium, Bacillus polymyxa, Bacilluspsychrosaccharolyticus, Bacillus pumilus, Bacillus sphaericus, Bacillussubtilis subsp. spizizenii, Bacillus subtilis subsp. subtilis orBacillus thuringiensis; Enterobacteriacae such as the generaCitrobacter, Edwardsiella, Enterobacter, Erwinia, Escherichia,Klebsiella, Salmonella or Serratia, for example the genera and speciesCitrobacter amalonaticus, Citrobacter diversus, Citrobacter freundii,Citrobacter genomospecies, Citrobacter gillenii, Citrobacterintermedium, Citrobacter koseri, Citrobacter murliniae, Citrobacter sp.,Edwardsiella hoshinae, Edwardsiella ictaluri, Edwardsiella tarda,Erwinia alni, Erwinia amylovora, Erwinia ananatis, Erwinia aphidicola,Erwinia billingiae, Erwinia cacticida, Erwinia cancerogena, Erwiniacarnegieana, Erwinia carotovora subsp. atroseptica, Erwinia carotovorasubsp. betavasculorum, Erwinia carotovora subsp. odorifera, Erwiniacarotovora subsp. wasabiae, Erwinia chrysanthemi, Erwinia cypripedii,Erwinia dissolvens, Erwinia herbicola, Erwinia mallotivora, Erwiniamilletiae, Erwinia nigrifluens, Erwinia nimipressuralis, Erwiniapersicina, Erwinia psidii, Erwinia pyrifoliae, Erwinia quercina, Erwiniarhapontici, Erwinia rubrifaciens, Erwinia salicis, Erwinia stewartii,Erwinia tracheiphila, Erwinia uredovora, Escherichia adecarboxylata,Escherichia anindolica, Escherichia aurescens, Escherichia blattae,Escherichia coli, Escherichia coli var. communior, Escherichiacoli-mutabile, Escherichia fergusonii, Escherichia hermannii,Escherichia sp., Escherichia vulneris, Klebsiella aerogenes, Klebsiellaedwardsii subsp. atlantae, Klebsiella ornithinolytica, Klebsiellaoxytoca, Klebsiella planticola, Klebsiella pneumoniae, Klebsiellapneumoniae subsp. pneumoniae, Klebsiella sp., Klebsiella terrigena,Klebsiella trevisanii, Salmonella abony, Salmonella arizonae, Salmonellabongori, Salmonella choleraesuis subsp. arizonae, Salmonellacholeraesuis subsp. bongori, Salmonella choleraesuis subsp.cholereasuis, Salmonella choleraesuis subsp. diarizonae, Salmonellacholeraesuis subsp. houtenae, Salmonella choleraesuis subsp. indica,Salmonella choleraesuis subsp. salamae, Salmonella daressalaam,Salmonella enterica subsp. houtenae, Salmonella enterica subsp. salamae,Salmonella enteritidis, Salmonella gallinarum, Salmonella heidelberg,Salmonella panama, Salmonella senftenberg, Salmonella typhimurium,Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratiagrimesii, Serratia liquefaciens, Serratia marcescens, Serratiamarcescens subsp. marcescens, Serratia marinorubra, Serratia odorifera,Serratia plymouthensis, Serratia plymuthica, Serratia proteamaculans,Serratia proteamaculans subsp. quinovora, Serratia quinivorans orSerratia rubidaea; Rhizobiaceae, such as the genera Agrobacterium,Carbophilus, Chelatobacter, Ensifer, Rhizobium, Sinorhizobium, forexample the genera and species Agrobacterium atlanticum, Agrobacteriumferrugineum, Agrobacterium gelatinovorum, Agrobacterium larrymoorei,Agrobacterium meteori, Agrobacterium radiobacter, Agrobacteriumrhizogenes, Agrobacterium rubi, Agrobacterium stellulatum, Agrobacteriumtumefaciens, Agrobacterium vitis, Carbophilus carboxidus, Chelatobacterheintzii, Ensifer adhaerens, Ensifer arboris, Ensifer fredii, Ensiferkostiensis, Ensifer kummerowiae, Ensifer medicae, Ensifer meliloti,Ensifer saheli, Ensifer terangae, Ensifer xinjiangensis, Rhizobiumciceri, Rhizobium etli, Rhizobium fredii, Rhizobium galegae, Rhizobiumgallicum, Rhizobium giardinii, Rhizobium hainanense, Rhizobium huakuii,Rhizobium huautlense, Rhizobium indigoferae, Rhizobium japonicum,Rhizobium leguminosarum, Rhizobium loessense, Rhizobium loti, Rhizobiumlupini, Rhizobium mediterraneum, Rhizobium meliloti, Rhizobiummongolense, Rhizobium phaseoli, Rhizobium radiobacter, Rhizobiumrhizogenes, Rhizobium rubi, Rhizobium sullae, Rhizobium tianshanense,Rhizobium trifolii, Rhizobium tropici, Rhizobium undicola, Rhizobiumvitis, Sinorhizobium adhaerens, Sinorhizobium arboris, Sinorhizobiumfredii, Sinorhizobium kostiense, Sinorhizobium kummerowiae,Sinorhizobium medicae, Sinorhizobium meliloti, Sinorhizobium morelense,Sinorhizobium saheli or Sinorhizobium xinjiangense.

Further utilizable host cells are detailed in: Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). Expression strains which can be used, for example those with alower protease activity, are described in: Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128. These include plant cells and certaintissues, organs and parts of plants in all their phenotypic forms suchas anthers, fibers, root hairs, stalks, embryos, calli, cotelydons,petioles, harvested material, plant tissue, reproductive tissue and cellcultures which are derived from the actual transgenic plant and/or canbe used for bringing about the transgenic plant. Polynucleotides orvectors can be introduced into the host cell as described above by meansof transformation or transfection methods which are known in the priorart. Conditions and media for the cultivation of the host cells are alsoknown to the skilled worker.

The host cell according to the invention preferably additionallycomprises at least one further enzyme which is involved in thebiosynthesis of lipids or fatty acids. Preferred enzymes have alreadybeen mentioned in another place in the description. The enzyme can bepresent in the host cell in endogenous form, i.e. the host cell alreadynaturally expresses a gene which codes for the enzyme in question.Alternatively, it is also possible to introduce, into the host cell, aheterologous polynucleotide which codes for the enzyme. Suitable methodsand means for the expression of a heterologous polynucleotide are knownin the prior art and are described herein in connection with thepolynucleotides, vectors and host cells according to the invention.

The invention also relates to a method of generating a polypeptide withdesaturase or elongase activity, comprising the steps:

-   -   (a) expressing a polynucleotide according to the invention in a        host cell; and    -   (b) obtaining, from the host cell, the polypeptide which is        encoded by the polynucleotide.

In this context, the polypeptide can be obtained by all current proteinpurification methods. The methods comprise, for example, affinitychromatography, molecular sieve chromatography, high-pressure liquidchromatography or else protein precipitation, if appropriate withspecific antibodies. Although this is preferred, the process need notnecessarily provide a pure polypeptide preparation.

The invention therefore also relates to a polypeptide which is encodedby the polynucleotide according to the invention or which is obtainableby the abovementioned method according to the invention.

The term “polypeptide” refers both to an essentially pure polypeptide,but also to a polypeptide preparation which additionally comprisesfurther components or impurities.

The term is also used for fusion proteins or protein aggregates whichcomprise the polypeptide according to the invention and additionallyfurther components. The term also refers to chemically modifiedpolypeptides. In this context, chemical modifications compriseartificial modifications or naturally occurring modifications, forexample posttranslational modifications such as phosphorylation,myristylation, glycosylation and the like. The terms polypeptide,peptide or protein are interchangeable and are used accordingly in thedescription and in the prior art. The polypeptides according to theinvention have the abovementioned biological activities, that is to saydesaturase or elongase activities, and can influence the biosynthesis ofpolyunsaturated fatty acids (PUFAs), preferably the long-chain PUFAs(LCPUFAs), as herein described.

The invention also comprises an antibody which specifically recognizesthe polypeptide according to the invention.

Antibodies against the polypeptide according to the invention can beprepared by means of known methods, where purified polypeptide orfragments thereof with suitable epitopes are used as the antigen.Suitable epitopes can be determined by means of known algorithms for theantigenicity determination, based on the amino acid sequences, of thepolypeptides according to the invention, provided herein. The relevantpolypeptides or fragments can then be synthesized or obtained byrecombinant techniques. After animals, preferably mammals, for examplehares, rats or mice, have been immunized, the antibodies can then beobtained from the serum, using known methods. Alternatively, monoclonalantibodies or antibody fragments can be provided with the known methods;see, for example, Harlow and Lane “Antibodies, A Laboratory Manual”, CSHPress, Cold Spring Harbor, 1988 or Köhler and Milstein, Nature 256(1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3.

The antibodies preferably take the form of monoclonal or polyclonalantibodies, single-chain antibodies or chimeric antibodies, andfragments of these such as Fab, Fv or scFv. Further antibodies withinthe meaning of the invention are bispecific antibodies, syntheticantibodies or their chemically modified derivatives.

It is intended that the antibodies according to the inventionspecifically recognize the polypeptides according to the invention, thatis to say they should not significantly cross-react with other proteins.This can be assayed by means of methods known in the prior art. Forexample, the antibodies can be employed for the purposes ofimmunoprecipitation, immunhistochemistry or protein purification (forexample affinity chromatography).

The invention furthermore relates to a transgenic, nonhuman organismwhich comprises the polynucleotide, the vector or the host cell of thepresent invention. The transgenic, nonhuman organism preferably takesthe form of an animal, a plant or a multicellular microorganism.

The term “transgenic” is understood as meaning that a heterologouspolynucleotide, that is to say a polynucleotide which does not occurnaturally in the respective organism, is introduced in the organism.This can be achieved either by random insertion of the polynucleotide orby homologous recombination. Naturally, it is also possible to introducethe vector according to the invention instead of the polynucleotide.Methods of introducing polynucleotides or vectors for the purposes ofrandom insertion or homologous recombination are known in the prior artand also described in greater detail hereinbelow. Host cells whichcomprise the polynucleotide or the vector can also be introduced into anorganism and thus generate a transgenic organism. In such a case, suchan organism takes the form of a chimeric organism, where only thosecells which are derived from the introduced cells are transgenic, i.e.comprise the heterologous polynucleotide.

The transgenic nonhuman organisms are preferably oil-producingorganisms, which means organisms which are used for the production ofoils, like fungi such as Mortierella or Thraustochytrium, algae such asNephroselmis, Pseudoscourfielda, Prasinococcus, Scherffelia,Tetraselmis, Mantoniella, Ostreococcus, Crypthecodinium, Phaeodactylumor plants.

Transgenic plants which can be used are, in principle, all plants, thatis to say both dicotyledonous and monocotyledonous plants. Theypreferably take the form of oil crop plants which comprise large amountsof lipid compounds, such as peanut, oilseed rape, canola, sunflower,safflower (Carthamus tinctoria), poppy, mustard, hemp, castor-oil plant,olive, sesame, Calendula, Punica, evening primrose, verbascum, thistle,wild roses, hazelnut, almond, macadamia, avocado, bay, pumpkin/squash,linseed, soybean, pistachios, borage, trees (oil palm, coconut orwalnut) or arable crops such as maize, wheat, rye, oats, triticale,rice, barley, cotton, cassava, pepper, Tagetes, Solanaceae plants suchas potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa orbushy plants (coffee, cacao, tea), Salix species, and perennial grassesand fodder crops. Preferred plants according to the invention are oilcrop plants such as peanut, oilseed rape, canola, sunflower, safflower,poppy, mustard, hemp, castor-oil plant, olive, Calendula, Punica,evening primrose, pumpkin/squash, linseed, soybean, borage, trees (oilpalm, coconut). Especially preferred are plants which are high in C18:2-and/or C18:3-fatty acids, such as sunflower, safflower, tobacco,verbascum, sesame, cotton, pumpkin/squash, poppy, evening primrose,walnut, linseed, hemp or thistle. Very especially preferred plants areplants such as safflower, sunflower, poppy, evening primrose, walnut,linseed or hemp. In principle, however, all plants which are capable ofsynthesizing fatty acids are suitable, such as all dicotyledonous ormonocotyledonous plants, algae or mosses. Advantageous plants areselected from the group of the plant families Adelotheciaceae,Anacardiaceae, Asteraceae, Apiaceae, Betulaceae, Boraginaceae,Brassicaceae, Bromeliaceae, Caricaceae, Cannabaceae, Convolvulaceae,Chenopodiaceae, Crypthecodiniaceae, Cucurbitaceae, Ditrichaceae,Elaeagnaceae, Ericaceae, Euphorbiaceae, Fabaceae, Geraniaceae,Gramineae, Juglandaceae, Lauraceae, Leguminosae, Linaceae,Prasinophyceae or vegetable plants or ornamentals such as Tagetes.

Examples which may be mentioned are the following plants selected fromthe group consisting of: Adelotheciaceae such as the generaPhyscomitrella, for example the genus and species Physcomitrella patens,Anacardiaceae such as the genera Pistacia, Mangifera, Anacardium, forexample the genus and species Pistacia vera [pistachio], Mangifer indica[mango] or Anacardium occidentale [cashew], Asteraceae, such as thegenera Calendula, Carthamus, Centaurea, Cichorium, Cynara, Helianthus,Lactuca, Locusta, Tagetes, Valeriana, for example the genus and speciesCalendula officinalis [common marigold], Carthamus tinctorius[safflower], Centaurea cyanus [cornflower], Cichorium intybus [chicory],Cynara scolymus [artichoke], Helianthus annus [sunflower], Lactucasativa, Lactuca crispa, Lactuca esculenta, Lactuca scariola L. ssp.sativa, Lactuca scariola L. var. integrata, Lactuca scariola L. var.integrifolia, Lactuca sativa subsp. romana, Locusta communis, Valerianalocusta [salad vegetables], Tagetes lucida, Tagetes erecta or Tagetestenuifolia [african or french marigold], Apiaceae, such as the genusDaucus, for example the genus and species Daucus carota [carrot],Betulaceae, such as the genus Corylus, for example the genera andspecies Corylus avellana or Corylus colurna [hazelnut], Boraginaceae,such as the genus Borago, for example the genus and species Boragoofficinalis [borage], Brassicaceae, such as the genera Brassica,Camelina, Melanosinapis, Sinapis, Arabadopsis, for example the generaand species Brassica napus, Brassica rapa ssp. [oilseed rape], Sinapisarvensis Brassica juncea, Brassica juncea var. juncea, Brassica junceavar. crispifolia, Brassica juncea var. foliosa, Brassica nigra, Brassicasinapioides, Camelina sativa, Melanosinapis communis [mustard], Brassicaoleracea [fodder beet] or Arabidopsis thaliana, Bromeliaceae, such asthe genera Anana, Bromelia (pineapple), for example the genera andspecies Anana comosus, Ananas ananas or Bromelia comosa [pineapple],Caricaceae, such as the genus Carica, such as the genus and speciesCarica papaya [pawpaw], Cannabaceae, such as the genus Cannabis, such asthe genus and species Cannabis sative [hemp], Convolvulaceae, such asthe genera Ipomea, Convolvulus, for example the genera and speciesIpomoea batatus, Ipomoea pandurata, Convolvulus batatas, Convolvulustiliaceus, Ipomoea fastigiata, Ipomoea tiliacea, Ipomoea triloba orConvolvulus panduratus [sweet potato, batate], Chenopodiaceae, such asthe genus Beta, such as the genera and species Beta vulgaris, Betavulgaris var. altissima, Beta vulgaris var. vulgaris, Beta maritima,Beta vulgaris var. perennis, Beta vulgaris var. conditiva or Betavulgaris var. esculenta [sugarbeet], Crypthecodiniaceae, such as thegenus Crypthecodinium, for example the genus and species Cryptecodiniumcohnii, Cucurbitaceae, such as the genus Cucurbita, for example thegenera and species Cucurbita maxima, Cucurbita mixta, Cucurbita pepo orCucurbita moschata [pumpkin/squash], Cymbellaceae, such as the generaAmphora, Cymbella, Okedenia, Phaeodactylum, Reimeria, for example thegenus and species Phaeodactylum tricornutum, Ditrichaceae, such as thegenera Ditrichaceae, Astomiopsis, Ceratodon, Chrysoblastella, Ditrichum,Distichium, Eccremidium, Lophidion, Philibertiella, Pleuridium,Saelania, Trichodon, Skottsbergia, for example the genera and speciesCeratodon antarcticus, Ceratodon columbiae, Ceratodon heterophyllus,Ceratodon purpurascens, Ceratodon purpureus, Ceratodon purpureus ssp.convolutus, Ceratodon purpureus ssp. stenocarpus, Ceratodon purpureusvar. rotundifolius, Ceratodon ratodon, Ceratodon stenocarpus,Chrysoblastella chilensis, Ditrichum ambiguum, Ditrichum brevisetum,Ditrichum crispatissimum, Ditrichum difficile, Ditrichum falcifolium,Ditrichum flexicaule, Ditrichum giganteum, Ditrichum heteromallum,Ditrichum lineare, Ditrichum montanum, Ditrichum montanum, Ditrichumpallidum, Ditrichum punctulatum, Ditrichum pusillum, Ditrichum pusillumvar. tortile, Ditrichum rhynchostegium, Ditrichum schimperi, Ditrichumtortile, Distichium capillaceum, Distichium hagenii, Distichiuminclinatum, Distichium macounii, Eccremidium floridanum, Eccremidiumwhiteleggei, Lophidion strictus, Pleuridium acuminatum, Pleuridiumalternifolium, Pleuridium holdridgei, Pleuridium mexicanum, Pleuridiumravenelii, Pleuridium subulatum, Saelania glaucescens, Trichodonborealis, Trichodon cylindricus or Trichodon cylindricus var. oblongus,Elaeagnaceae, such as the genus Elaeagnus, for example the genus andspecies Olea europaea [olive], Ericaceae, such as the genus Kalmia, forexample the genera and species Kalmia latifolia, Kalmia angustifolia,Kalmia microphylla, Kalmia polifolia, Kalmia occidentalis, Cistuschamaerhodendros or Kalmia lucida [mountain laurel], Euphorbiaceae, suchas the genera Manihot, Janipha, Jatropha, Ricinus, for example thegenera and species Manihot utilissima, Janipha manihot, Jatrophamanihot, Manihot aipil, Manihot dulcis, Manihot manihot, Manihotmelanobasis, Manihot esculenta [cassava] or Ricinus communis [castor-oilplant], Fabaceae, such as the genera Pisum, Albizia, Cathormion,Feuillea, Inga, Pithecolobium, Acacia, Mimosa, Medicajo, Glycine,Dolichos, Phaseolus, soybean, for example the genera and species Pisumsativum, Pisum arvense, Pisum humile [pea], Albizia berteriana, Albiziajulibrissin, Albizia lebbeck, Acacia berteriana, Acacia littoralis,Albizia berteriana, Albizzia berteriana, Cathormion berteriana, Feuilleaberteriana, Inga fragrans, Pithecellobium berterianum, Pithecellobiumfragrans, Pithecolobium berterianum, Pseudalbizzia berteriana, Acaciajulibrissin, Acacia nemu, Albizia nemu, Feuilleea julibrissin, Mimosajulibrissin, Mimosa speciosa, Sericanrda julibrissin, Acacia lebbeck,Acacia macrophylla, Albizia lebbeck, Feuilleea lebbeck, Mimosa lebbeck,Mimosa speciosa [silk tree], Medicago sativa, Medicago falcata, Medicagovaria [alfalfa]Glycine max Dolichos soja, Glycine gracilis, Glycinehispida, Phaseolus max, Soja hispida or Soja max [soybean], Funariaceae,such as the genera Aphanorrhegma, Entosthodon, Funaria, Physcomitrella,Physcomitrium, for example the genera and species Aphanorrhegmaserratum, Entosthodon attenuatus, Entosthodon bolanderi, Entosthodonbonplandii, Entosthodon californicus, Entosthodon drummondii,Entosthodon jamesonii, Entosthodon leibergii, Entosthodon neoscoticus,Entosthodon rubrisetus, Entosthodon spathulifolius, Entosthodon tucsoni,Funaria americana, Funaria bolanderi, Funaria calcarea, Funariacalifornica, Funaria calvescens, Funaria convoluta, Funaria flavicans,Funaria groutiana, Funaria hygrometrica, Funaria hygrometrica var.arctica, Funaria hygrometrica var. calvescens, Funaria hygrometrica var.convoluta, Funaria hygrometrica var. muralis, Funaria hygrometrica var.utahensis, Funaria microstoma, Funaria microstoma var. obtusifolia,Funaria muhlenbergii, Funaria orcuttii, Funaria plano-convexa, Funariapolaris, Funaria ravenelii, Funaria rubriseta, Funaria serrata, Funariasonorae, Funaria sublimbatus, Funaria tucsoni, Physcomitrellacalifornica, Physcomitrella patens, Physcomitrella readeri,Physcomitrium australe, Physcomitrium californicum, Physcomitriumcollenchymatum, Physcomitrium coloradense, Physcomitrium cupuliferum,Physcomitrium drummondii, Physcomitrium eurystomum, Physcomitriumflexifolium, Physcomitrium hookeri, Physcomitrium hookeri var. serratum,Physcomitrium immersum, Physcomitrium kellermanii, Physcomitriummegalocarpum, Physcomitrium pyriforme, Physcomitrium pyriforme var.serratum, Physcomitrium rufipes, Physcomitrium sandbergii, Physcomitriumsubsphaericum, Physcomitrium washingtoniense, Geraniaceae, such as thegenera Pelargonium, Cocos, Oleum, for example the genera and speciesCocos nucifera, Pelargonium grossularioides or Oleum cocois [coconut],Gramineae, such as the genus Saccharum, for example the genus andspecies Saccharum officinarum, Juglandaceae, such as the genera Juglans,Wallia, for example the genera and species Juglans regia, Juglansailanthifolia, Juglans sieboldiana, Juglans cinerea, Wallia cinerea,Juglans bixbyi, Juglans californica, Juglans hindsii, Juglansintermedia, Juglans jamaicensis, Juglans major, Juglans microcarpa,Juglans nigra or Wallia nigra [walnut], Lauraceae, such as the generaPersea, Laurus, for example the genera and species Laurus nobilis [bay],Persea americana, Persea gratissima or Persea persea [avocado],Leguminosae, such as the genus Arachis, for example the genus andspecies Arachis hypogaea [peanut], Linaceae, such as the genera Linum,Adenolinum, for example the genera and species Linum usitatissimum,Linum humile, Linum austriacum, Linum bienne, Linum angustifolium, Linumcatharticum, Linum flavum, Linum grandiflorum, Adenolinum grandiflorum,Linum lewisii, Linum narbonense, Linum perenne, Linum perenne var.lewisii, Linum pratense or Linum trigynum [linseed], Lythrarieae, suchas the genus Punica, for example the genus and species Punica granatum[pomegranate], Malvaceae, such as the genus Gossypium, for example thegenera and species Gossypium hirsutum, Gossypium arboreum, Gossypiumbarbadense, Gossypium herbaceum or Gossypium thurberi [cotton],Marchantiaceae, such as the genus Marchantia, for example the genera andspecies Marchantia berteroana, Marchantia foliacea, Marchantiamacropora, Musaceae, such as the genus Musa, for example the genera andspecies Musa nana, Musa acuminata, Musa paradisiaca, Musa spp. [banana],Onagraceae, such as the genera Camissonia, Oenothera, for example thegenera and species Oenothera biennis or Camissonia brevipes [eveningprimrose], Palmae, such as the genus Elacis, for example the genus andspecies Elaeis guineensis [oil palm], Papaveraceae, such as, forexample, the genus Papaver, for example the genera and species Papaverorientale, Papaver rhoeas, Papaver dubium [poppy], Pedaliaceae, such asthe genus Sesamum, for example the genus and species Sesamum indicum[sesame], Piperaceae, such as the genera Piper, Artanthe, Peperomia,Steffensia, for example the genera and species Piper aduncum, Piperamalago, Piper angustifolium, Piper auritum, Piper betel, Piper cubeba,Piper longum, Piper nigrum, Piper retrofractum, Artanthe adunca,Artanthe elongata, Peperomia elongata, Piper elongatum, Steffensiaelongata [cayenne pepper], Poaceae, such as the genera Hordeum, Secale,Avena, Sorghum, Andropogon, Holcus, Panicum, Oryza, Zea (maize),Triticum, for example the genera and species Hordeum vulgare, Hordeumjubatum, Hordeum murinum, Hordeum secalinum, Hordeum distichon, Hordeumaegiceras, Hordeum hexastichon, Hordeum hexastichum, Hordeum irregulare,Hordeum sativum, Hordeum secalinum [barley], Secale cereale [rye], Avenasativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avenahybrida [oats], Sorghum bicolor, Sorghum halepense, Sorghum saccharatum,Sorghum vulgare, Andropogon drummondii, Holcus bicolor, Holcus sorghum,Sorghum aethiopicum, Sorghum arundinaceum, Sorghum caffrorum, Sorghumcernuum, Sorghum dochna, Sorghum drummondii, Sorghum durra, Sorghumguineense, Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum,Sorghum subglabrescens, Sorghum verticilliflorum, Sorghum vulgare,Holcus halepensis, Sorghum miliaceum, Panicum militaceum [millet], Oryzasativa, Oryza latifolia [rice], Zea mays [maize]Triticum aestivum,Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha,Triticum sativum or Triticum vulgare [wheat], Porphyridiaceae, such asthe genera Chroothece, Flintiella, Petrovanella, Porphyridium, Rhodella,Rhodosorus, Vanhoeffenia, for example the genus and species Porphyridiumcruentum, Proteaceae, such as the genus Macadamia, for example the genusand species Macadamia intergrifolia [macadamia], Prasinophyceae, such asthe genera Nephroselmis, Prasinococcus, Scherffelia, Tetraselmis,Mantoniella, Ostreococcus, for example the genera and speciesNephroselmis olivacea, Prasinococcus capsulatus, Scherffelia dubia,Tetraselmis chui, Tetraselmis suecica, Mantoniella squamata,Ostreococcus tauri, Rubiaceae, such as the genus Coffea, for example thegenera and species Cofea spp., Coffea arabica, Coffea canephora orCoffea liberica [coffee], Scrophulariaceae, such as the genus Verbascum,for example the genera and species Verbascum blattaria, Verbascumchaixii, Verbascum densiflorum, Verbascum lagurus, Verbascumlongifolium, Verbascum lychnitis, Verbascum nigrum, Verbascum olympicum,Verbascum phlomoides, Verbascum phoenicum, Verbascum pulverulentum orVerbascum thapsus [verbascum], Solanaceae, such as the genera Capsicum,Nicotiana, Solanum, Lycopersicon, for example the genera and speciesCapsicum annuum, Capsicum annuum var. glabriusculum, Capsicum frutescens[pepper], Capsicum annuum [paprika], Nicotiana tabacum, Nicotiana alata,Nicotiana attenuata, Nicotiana glauca, Nicotiana langsdorffii, Nicotianaobtusifolia, Nicotiana quadrivalvis, Nicotiana repanda, Nicotianarustica, Nicotiana sylvestris [tobacco], Solanum tuberosum [potato],Solanum melongena [eggplant], Lycopersicon esculentum, Lycopersiconlycopersicum, Lycopersicon pyriforme, Solanum integrifolium or Solanumlycopersicum [tomato], Sterculiaceae, such as the genus Theobroma, forexample the genus and species Theobroma cacao [cacao] or Theaceae, suchas the genus Camellia, for example the genus and species Camelliasinensis [tea].

Multicellular microorganisms which can be employed as transgenicnonhuman organisms are preferably protists or diatoms selected from thegroup of the families Dinophyceae, Turaniellidae or Oxytrichidae, suchas the genera and species: Crypthecodinium cohnii, Phaeodactylumtricornutum, Stylonychia mytilus, Stylonychia pustulata, Stylonychiaputrina, Stylonychia notophora, Stylonychia sp., Colpidium campylum orColpidium sp.

The invention relates to a process for the production of a substancewhich has the structure shown in the general formula I hereinbelow

the variables and substituents being the following:

-   -   R¹=hydroxyl, coenzyme A (thioester), lysophosphatidylcholine,        lysophosphatidylethanolamine, lysophosphatidylglycerol,        lyso-diphosphatidylglycerol, lysophosphatidylserine,        lysophosphatidylinositol, sphingo base or a radical of the        formula II

-   -   R²=hydrogen, lysophosphatidylcholine,        lysophosphatidylethanolamine, lysophosphatidylglycerol,        lysodiphosphatidylglycerol, lysophosphatidyl-serine,        lysophosphatidylinositol or saturated or unsaturated        C₂-C₂₄-alkylcarbonyl,    -   R³=hydrogen, saturated or unsaturated C₂-C₂₄-alkylcarbonyl, or        R² and R³ independently of one another are a radical of the        formula Ia:

-   -   in which    -   n=2, 3, 4, 5, 6, 7 or 9, m=2, 3, 4, 5 or 6 and p=0 or 3. and        where the process comprises the cultivation of (i) a host cell        according to the invention or (ii) a transgenic nonhuman        according to the invention under conditions which permit the        biosynthesis of the substance. Preferably, the abovementioned        substance is provided in an amount of at least 1% by weight        based on the total lipid content in the host cell or the        transgenic organism.

R¹ in the general formula I is hydroxyl, coenzyme A (thioester),lysophosphatidylcholine, lysophosphatidylethanolamine,lysophosphatidylglycerol, lyso-diphosphatidylglycerol,lysophosphatidylserine, lysophosphatidylinositol, sphingo base or aradical of the general formula II

The abovementioned radicals of R¹ are always bonded to the compounds ofthe general formula I in the form of their thioesters.

R² in the general formula II is hydrogen, lysophosphatidylcholine,lysophosphatidylethanolamine, lysophosphatidylglycerol,lysodiphosphatidylglycerol, lysophosphatidylserine,lysophosphatidylinositol or saturated or unsaturatedC₂-C₂₄-alkylcarbonyl.

Alkyl radicals which may be mentioned are substituted or unsubstituted,saturated or unsaturated C₂-C₂₄-alkylcarbonyl chains such asethylcarbonyl, n-propylcarbonyl, n-butylcarbonyl, n-pentylcarbonyl,n-hexylcarbonyl, n-heptylcarbonyl, n-octylcarbonyl, n-nonylcarbonyl,n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl,n-tridecyl-carbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl,n-hexadecylcarbonyl, n-hepta-decylcarbonyl, n-octadecylcarbonyl,n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl orn-tetracosanylcarbonyl, which comprise one or more double bonds.Saturated or unsaturated C₁₀-C₂₂-alkylcarbonyl radicals such asn-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl,n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl,n-hexadecylcarbonyl, n-heptadecylcarbonyl, n-octadecylcarbonyl,n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl orn-tetracosanylcarbonyl, which comprise one or more double bonds arepreferred. Especially preferred are saturated and/or unsaturatedC₁₀-C₂₂-alkylcarbonyl radicals such as C₁₀-alkylcarbonyl,C₁₁-alkylcarbonyl, C₁₂-alkylcarbonyl, C₁₃-alkylcarbonyl,C₁₄-alkylcarbonyl, C₁₆-alkylcarbonyl, C₁₈-alkylcarbonyl,C₂₀-alkylcarbonyl or C₂₂-alkylcarbonyl radicals which comprise one ormore double bonds. Very especially preferred are saturated orunsaturated C₁₆-C₂₂-alkylcarbonyl radicals such as C₁₆-alkylcarbonyl,C₁₈-alkylcarbonyl, C₂₀-alkylcarbonyl or C₂₂-alkylcarbonyl radicals whichcomprise one or more double bonds. These advantageous radicals cancomprise two, three, four, five or six double bonds. The especiallyadvantageous radicals with 20 or 22 carbon atoms in the fatty acid chaincomprise up to six double bonds, advantageously three, four, five or sixdouble bonds, especially preferably five or six double bonds. All theabovementioned radicals are derived from the corresponding fatty acids.

R³ in the general formula II is hydrogen, saturated or unsaturatedC₂-C₂₄-alkylcarbonyl.

Alkyl radicals which may be mentioned are substituted or unsubstituted,saturated or unsaturated C₂-C₂₄-alkylcarbonyl chains such asethylcarbonyl, n-propylcarbonyl, n-butylcarbonyl-, n-pentylcarbonyl,n-hexylcarbonyl, n-heptylcarbonyl, n-octylcarbonyl, n-nonylcarbonyl,n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl,n-tridecyl-carbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl,n-hexadecylcarbonyl, n-hepta-decylcarbonyl, n-octadecylcarbonyl-,n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl orn-tetracosanylcarbonyl, which comprise one or more double bonds.Saturated or unsaturated C₁₀-C₂₂-alkylcarbonyl radicals such asn-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl,n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl,n-hexadecylcarbonyl, n-heptadecylcarbonyl, n-octadecylcarbonyl,n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl orn-tetracosanylcarbonyl, which comprise one or more double bonds arepreferred. Especially preferred are saturated and/or unsaturatedC₁₀-C₂₂-alkylcarbonyl radicals such as C₁₀-alkylcarbonyl,C₁₁-alkylcarbonyl, C₁₂-alkylcarbonyl, C₁₃-alkylcarbonyl,C₁₄-alkylcarbonyl, C₁₆-alkylcarbonyl, C₁₈-alkylcarbonyl,C₂₀-alkylcarbonyl or C₂₂-alkylcarbonyl radicals which comprise one ormore double bonds. Very especially preferred are saturated orunsaturated C₁₆-C₂₂-alkylcarbonyl radicals such as C₁₆-alkylcarbonyl,C₁₈-alkylcarbonyl, C₂₀-alkylcarbonyl or C₂₂-alkylcarbonyl radicals whichcomprise one or more double bonds. These advantageous radicals cancomprise two, three, four, five or six double bonds. The especiallyadvantageous radicals with 20 or 22 carbon atoms in the fatty acid chaincomprise up to six double bonds, advantageously three, four, five or sixdouble bonds, especially preferably five or six double bonds. All theabovementioned radicals are derived from the corresponding fatty acids.

The abovementioned radicals of R¹, R² and R³ can be substituted byhydroxyl and/or epoxy groups and/or can comprise triple bonds.

The polyunsaturated fatty acids produced in the process according to theinvention advantageously comprise at least two, advantageously three,four, five or six, double bonds. The fatty acids especiallyadvantageously comprise four, five or six double bonds. Fatty acidsproduced in the process advantageously have 18, 20 or 22 C atoms in thefatty acid chain; the fatty acids preferably comprise 20 or 22 carbonatoms in the fatty acid chain. Saturated fatty acids are advantageouslyreacted to a minor degree, or not at all, with the nucleic acids used inthe process. To a minor degree is to be understood as meaning that thesaturated fatty acids are reacted with less than 5% of the activity,advantageously less than 3%, especially advantageously with less than2%, very especially preferably with less than 1, 0.5, 0.25 or 0.125% incomparison with polyunsaturated fatty acids. These fatty acids whichhave been produced can be produced in the process as a single product orbe present in a fatty acid mixture.

Advantageously, the substituents R² or R³ in the general formulae I andII are, independently of one another, saturated or unsaturatedC₁₈-C₂₂-alkylcarbonyl, especially advantageously, they are,independently of one another, unsaturated C₁₈-, C₂₀- orC₂₂-alkylcarbonyl with at least two double bonds.

The polyunsaturated fatty acids produced in the process areadvantageously bound in membrane lipids and/or triacylglycerides, butmay also occur in the organisms as free fatty acids or else bound in theform of other fatty acid esters. In this context, they may be present as“pure products” or else advantageously in the form of mixtures ofvarious fatty acids or mixtures of different glycerides. The variousfatty acids which are bound in the triacylglycerides can be derived fromshort-chain fatty acids with 4 to 6 C atoms, medium-chain fatty acidswith 8 to 12 C atoms or long-chain fatty acids with 14 to 24 C atoms;preferred are long-chain fatty acids, more preferably long-chainpolyunsaturated fatty acids with 18, 20 and/or 22 C atoms.

The process according to the invention advantageously yields fatty acidesters with polyunsaturated C₁₈-, C₂₀- and/or C₂₂-fatty acid moleculeswith at least two double bonds in the fatty acid ester, advantageouslywith at least three, four, five or six double bonds in the fatty acidester, especially advantageously with at least five or six double bondsin the fatty acid ester and advantageously leads to the synthesis oflinoleic acid (=LA, C18:^(2Δ9,12)), γ-linolenic acid (=GLA,C18:3^(Δ6,9,12)), stearidonic acid (=SDA, C18:4^(Δ6,9,12,15)),dihomo-γ-linolenic acid (=DGLA, 20:3^(Δ8,11,14)), ω3-eicosatetraenoicacid (=ETA, C20:4^(Δ5,8,11,14)), arachidonic acid (ARA,C20:4^(Δ5,8,11,14)), eicosapentaenoic acid (EPA, C20:5^(Δ5,8,11,14,17)),ω6-docosapentaenoic acid (C22:5^(Δ4,7,10,13,16)), ω6-docosatetraenoicacid (C22:4^(Δ7,10,13,16)), ω3-docosapentaenoic acid (=DPA,C22:5^(Δ7,10,13,16,19)), docosahexaenoic acid (=DHA,C22:6^(Δ4,7,10,13,16,19)) or mixtures of these, preferably ARA, EPAand/or DHA. ω3-Fatty acids such as EPA and/or DHA are very especiallypreferably produced.

The fatty acid esters with polyunsaturated C₁₈-, C₂₀- and/or C₂₂-fattyacid molecules can be isolated in the form of an oil or lipid, forexample in the form of compounds such as sphingolipids,phosphoglycerides, lipids, glycolipids such as glycosphingolipids,phospholipids such as phosphatidylethanolamine, phosphatidylcholine,phosphatidylserine, phosphatidylglycerol, phosphatidylinositol ordiphosphatidylglycerol, monoacylglycerides, diacylglycerides,triacylglycerides or other fatty acid esters such as the acetyl-coenzymeA esters which comprise the polyunsaturated fatty acids with at leasttwo, three, four, five or six, preferably five or six double bonds, fromthe organisms which have been used for the preparation of the fatty acidesters; advantageously, they are isolated in the form of theirdiacylglycerides, triacylglycerides and/or in the form ofphosphatidylcholine, especially preferably in the form of thetriacylglycerides. In addition to these esters, the polyunsaturatedfatty acids are also present in the organisms, advantageously theplants, as free fatty acids or bound in other compounds. As a rule, thevarious abovementioned compounds (fatty acid esters and free fattyacids) are present in the organisms with an approximate distribution of80 to 90% by weight of triglycerides, 2 to 5% by weight of diglycerides,5 to 10% by weight of monoglycerides, 1 to 5% by weight of free fattyacids, 2 to 8% by weight of phospholipids, the total of the variouscompounds amounting to 100% by weight.

The process according to the invention yields the LCPUFAs produced in acontent of at least 3% by weight, advantageously at least 5% by weight,preferably at least 8% by weight, especially preferably at least 10% byweight, most preferably at least 15% by weight, based on the total fattyacids in the transgenic organisms, advantageously in a transgenic plant.In this context, it is advantageous to convert C₁₈- and/or C₂₀-fattyacids which are present in the host organisms to at least 10%,advantageously to at least 20%, especially advantageously to at least30%, most advantageously to at least 40% to give the correspondingproducts such as DPA or DHA, to mention just two examples. The fattyacids are advantageously produced in bound form. These unsaturated fattyacids can, with the aid of the nucleic acids used in the processaccording to the invention, be positioned at the sn1, sn2 and/or sn3position of the advantageously produced triglycerides. Since a pluralityof reaction steps are performed by the starting compounds linoleic acid(C18:2) and linolenic acid (C18:3) in the process according to theinvention, the end products of the process such as, for example,arachidonic acid (ARA), eicosapentaenoic acid (EPA), ω6-docosapentaenoicacid or DHA are not obtained as absolutely pure products; minor tracesof the precursors are always present in the end product. If, forexample, both linoleic acid and linolenic acid are present in thestarting organism and the starting plant, the end products such as ARA,EPA or DHA are present as mixtures. The precursors should advantageouslynot amount to more than 20% by weight, preferably not to more than 15%by weight, especially preferably not to more than 10% by weight, mostpreferably not to more than 5% by weight, based on the amount of the endproduct in question.

Advantageously, only ARA, EPA or only DHA, bound or as free acids, areproduced as end products in a transgenic plant in the process accordingto the invention. If the compounds ARA, EPA and DHA are producedsimultaneously, they are advantageously produced in a ratio of at least1:1:2 (EPA:ARA:DHA), advantageously of at least 1:1:3, preferably 1:1:4,especially preferably 1:1:5.

Fatty acid esters or fatty acid mixtures produced by the processaccording to the invention advantageously comprise 6 to 15% of palmiticacid, 1 to 6% of stearic acid, 7-85% of oleic acid, 0.5 to 8% ofvaccenic acid, 0.1 to 1% of arachic acid, 7 to 25% of saturated fattyacids, 8 to 85% of monounsaturated fatty acids and 60 to 85% ofpolyunsaturated fatty acids, in each case based on 100% and on the totalfatty acid content of the organisms. Advantageous polyunsaturated fattyacids which are present in the fatty acid esters or fatty acid mixturesare preferably at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or1% of arachidonic acid, based on the total fatty acid content.

Moreover, the fatty acid esters or fatty acid mixtures which have beenproduced by the process of the invention advantageously comprise fattyacids selected from the group of the fatty acids erucic acid(13-docosaenoic acid), sterculic acid (9,10-methyleneoctadec-9-enoicacid), malvalic acid (8,9-methyleneheptadec-8-enoic acid), chaulmoogricacid (cyclopentenedodecanoic acid), furan fatty acid(9,12-epoxyoctadeca-9,11-dienoic acid), vernolic acid(9,10-epoxyoctadec-12-enoic acid), tariric acid (6-octadecynoic acid),6-nonadecynoic acid, santalbic acid (t11-octadecen-9-ynoic acid),6,9-octadecenynoic acid, pyrulic acid (t10-heptadecen-8-ynoic acid),crepenynic acid (9-octadecen-12-ynoic acid), 13,14-dihydrooropheic acid,octadecen-13-ene-9,11-diynoic acid, petroselenic acid(cis-6-octadecenoic acid), 9c,12t-octadecadienoic acid, calendulic acid(8t10t12c-octadecatrienoic acid), catalpic acid(9t11t13c-octadecatrienoic acid), eleostearic acid(9c11t13t-octadecatrienoic acid), jacaric acid(8c10t12c-octadecatrienoic acid), punicic acid(9c11t13c-octadecatrienoic acid), parinaric acid(9c11t13t15c-octadecatetraenoic acid), pinolenic acid(all-cis-5,9,12-octadecatrienoic acid), laballenic acid(5,6-octadecadienallenic acid), ricinoleic acid (12-hydroxyoleic acid)and/or coriolic acid (13-hydroxy-9c,11t-octadecadienoic acid). Theabovementioned fatty acids are, as a rule, advantageously only found intraces in the fatty acid esters or fatty acid mixtures produced by theprocess according to the invention, that is to say that, based on thetotal fatty acids, they occur to less than 30%, preferably to less than25%, 24%, 23%, 22% or 21%, especially preferably to less than 20%, 15%,10%, 9%, 8%, 7%, 6% or 5%, very especially preferably to less than 4%,3%, 2% or 1%. The fatty acid esters or fatty acid mixtures produced bythe process according to the invention advantageously comprise less than0.1%, based on the total fatty acids, or no butyric acid, nocholesterol, no clupanodonic acid (=docosapentaenoic acid,C22:5^(Δ4,8,12,15,21)) and no nisinic acid (tetracosahexaenoic acid,C23:6^(Δ3,8,12,15,18,21)). Owing to the nucleic acid sequences of theinvention, or the nucleic acid sequences used in the process accordingto the invention, an increase in the yield of polyunsaturated fattyacids of at least 50%, advantageously of at least 80%, especiallyadvantageously of at least 100%, very especially advantageously of atleast 150%, in comparison with the nontransgenic starting organism, forexample a yeast, an alga, a fungus or a plant such as Arabidopsis orlinseed can be obtained when the fatty acids are detected by GC analysis(see examples).

Chemically pure polyunsaturated fatty acids or fatty acid compositionscan also be prepared by the processes described above. To this end, thefatty acids or the fatty acid compositions are isolated from theorganism, such as the microorganisms or the plants or the culture mediumin or on which the organisms have been grown, or from the organism andthe culture medium, in the known manner, for example via extraction,distillation, crystallization, chromatography or a combination of thesemethods. These chemically pure fatty acids or fatty acid compositionsare advantageous for applications in the food industry sector, thecosmetic industry sector and especially the pharmacological industrysector.

In principle, all genes of the fatty acid or lipid metabolism can beused in the process for the production of polyunsaturated fatty acids,advantageously in combination with the inventive polynucleotide(s) [forthe purposes of the present invention, the plural is understood asencompassing the singular and vice versa]. Genes of the fatty acid orlipid metabolism which are used are advantageously selected from thegroup consisting of acyl-CoA dehydrogenase(s), acyl-ACP [=acyl carrierprotein]desaturase(s), acyl-ACP thioesterase(s), fatty acidacyltransferase(s), acyl-CoA:lysophospholipid acyltransferases, fattyacid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme Acarboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s),fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, alleneoxide synthases, hydroperoxide lyases or fatty acid elongase(s). Genesselected from the group of the Δ4-desaturases, Δ5-desaturases,Δ6-desaturases, Δ9-desaturases, Δ12-desaturases, Δ6-elongases orΔ5-elongases in combination with the polynucleotides according to theinvention are particularly preferably used, it being possible to useindividual genes or a plurality of genes in combination.

Advantageously, the desaturases used in the process according to theinvention convert their respective substrates in the form of theCoA-fatty acid esters. If preceded by an elongation step, thisadvantageously results in an increased product yield. The respectivedesaturation products are thereby synthesized in greater quantities,since the elongation step is usually carried out with the CoA-fatty acidesters, while the desaturation step is predominantly carried out withthe phospholipids or the triglycerides. Therefore, a substitutionreaction between the CoA-fatty acid esters and the phospholipids ortriglycerides, which would require a further, possibly limiting, enzymereaction, is not necessary.

Owing to the enzymatic activity of the polypeptides used in the processaccording to the invention, a wide range of polyunsaturated fatty acidscan be produced in the process according to the invention. Depending onthe choice of the organisms, such as the advantageous plants, used forthe process according to the invention, mixtures of the variouspolyunsaturated fatty acids or individual polyunsaturated fatty acids,such as EPA or ARA, can be produced in free or bound form. Depending onthe prevailing fatty acid composition in the starting plant (C18:2- orC18:3-fatty acids), fatty acids which are derived from C18:2-fattyacids, such as GLA, DGLA or ARA, or fatty acids which are derived fromC18:3-fatty acids, such as SDA, ETA or EPA, are thus obtained. If onlylinoleic acid (=LA, C18:2^(Δ9,12)) is present as unsaturated fatty acidin the plant used for the process, the process can only afford GLA, DGLAand ARA as products, all of which can be present as free fatty acids orin bound form. If only α-linolenic acid (=ALA, C18:3^(Δ9,12,15)) ispresent as unsaturated fatty acid in the plant used for the process, asis the case, for example, in linseed, the process can only afford SDA,ETA, EPA and/or DHA as products, all of which can be present as freefatty acids or in bound form, as described above. Owing to themodification of the activity of the enzymes Δ5-desaturase,Δ6-desaturase, Δ4-desaturase, Δ12-desaturase, Δ5-elongase and/orΔ6-elongase which play a role in the synthesis, it is possible toproduce, in a targeted fashion, only individual products in theabovementioned organisms, advantageously in the abovementioned plants.Owing to the activity of Δ6-desaturase and Δ6-elongase, for example, GLAand DGLA, or SDA and ETA, are formed, depending on the starting plantand unsaturated fatty acid. DGLA or ETA or mixtures of these arepreferably formed. If Δ5-desaturase, Δ5-elongase and Δ4-desaturase areadditionally introduced into the organisms, advantageously into theplant, ARA, EPA and/or DHA are additionally formed. Advantageously, onlyARA, EPA or DHA or mixtures of these are synthesized, depending on thefatty acid present in the organism, or in the plant, which acts asstarting substance for the synthesis. Since biosynthetic cascades areinvolved, the end products in question are not present in puresubstances in the organisms. Small amounts of the precursor compoundsare always additionally present in the end product. These small amountsamount to less than 20% by weight, advantageously less than 15% byweight, especially advantageously less than 10% by weight, mostadvantageously less than 5, 4, 3, 2 or 1% by weight, based on the endproduct DGLA, ETA or their mixtures, or ARA, EPA, DHA or their mixtures,advantageously EPA or DHA or their mixtures.

In addition to the production, directly in the organism, of the startingfatty acids for the polypeptides used in the process of the invention,the fatty acids can also be fed externally. The production in theorganism is preferred for reasons of economy. Preferred substrates arelinoleic acid (C18:2^(Δ9,12)), γ-linolenic acid (C18:3^(Δ6,9,12)),eicosadienoic acid (C20:2^(Δ11,14)), dihomo-γ-linolenic acid(C20:3^(Δ8,11,14)), arachidonic acid (C20:4^(Δ5,8,11,14)),docosatetraenoic acid (C22:4^(Δ7,10,13,16)) and docosapentaenoic acid(C22:5^(Δ4,7,10,13,15)).

To increase the yield in the above-described process for the productionof oils and/or triglycerides with an advantageously elevated content ofpolyunsaturated fatty acids, it is advantageous to increase the amountof starting product for the synthesis of fatty acids; this can beachieved for example by introducing, into the organism, a nucleic acidwhich encodes a polypeptide with Δ12-desaturase. This is particularlyadvantageous in oil-producing organisms such as those from the family ofthe Brassicaceae, such as the genus Brassica, for example oilseed rape;the family of the Elaeagnaceae, such as the genus Elaeagnus, for examplethe genus and species Olea europaea, or the family Fabaceae, such as thegenus Glycine, for example the genus and species Glycine max, which arehigh in oleic acid. Since these organisms are only low in linoleic acid(Mikoklajczak et al., Journal of the American Oil Chemical Society, 38,1961, 678-681), the use of the abovementioned Δ12-desaturases forproducing the starting product linoleic acid is advantageous.

The process according to the invention advantageously employs theabovementioned nucleic acid sequences or their derivatives or homologueswhich code for polypeptides which retain the enzymatic activity of theproteins encoded by nucleic acid sequences. These sequences,individually or in combination with the polynucleotides according to theinvention, are cloned into expression constructs and used for theintroduction into, and expression in, organisms. Owing to theirconstruction, these expression constructs make possible an advantageousoptimal synthesis of the polyunsaturated fatty acids produced in theprocess according to the invention.

In a preferred embodiment, the process furthermore comprises the step ofobtaining a cell or an intact organism which comprises the nucleic acidsequences used in the process, where the cell and/or the organism istransformed with a polynucleotide according to the invention, a geneconstruct or a vector as described below, alone or in combination withfurther nucleic acid sequences which code for proteins of the fatty acidor lipid metabolism. In a further preferred embodiment, this processfurthermore comprises the step of obtaining the oils, lipids or freefatty acids from the organism or from the culture. The culture can, forexample, take the form of a fermentation culture, for example in thecase of the cultivation of microorganisms, such as, for example,Mortierella, Thalassiosira, Mantoniella, Ostreococcus, Saccharomyces orThraustochytrium, or a greenhouse- or field-grown culture of a plant.The cell or the organism thus produced is advantageously a cell of anoil-producing organism, such as an oil crop, such as, for example,peanut, oilseed rape, canola, linseed, hemp, soybean, safflower,sunflowers or borage.

In the case of plant cells, plant tissue or plant organs, “growing” isunderstood as meaning, for example, the cultivation on or in a nutrientmedium, or of the intact plant on or in a substrate, for example in ahydroponic culture, potting compost or on arable land.

Suitable organisms or host cells for the process according to theinvention are those which are capable of synthesizing fatty acids,specifically unsaturated fatty acids, and/or which are suitable for theexpression of recombinant genes. Examples which may be mentioned areplants such as Arabidopsis, Asteraceae such as Calendula or crop plantssuch as soybean, peanut, castor-oil plant, sunflower, maize, cotton,flax, oilseed rape, coconut, oil palm, safflower (Carthamus tinctorius)or cacao bean, microorganisms, such as fungi, for example the genusMortierella, Thraustochytrium, Saprolegnia, Phytophthora or Pythium,bacteria, such as the genus Escherichia or Shewanella, yeasts, such asthe genus Saccharomyces, cyanobacteria, ciliates, algae such asMantoniella or Ostreococcus, or protozoans such as dinoflagellates, suchas Thalassiosira or Crypthecodinium. Preferred organisms are those whichare naturally capable of synthesizing substantial amounts of oil, suchas fungi, such as Mortierella alpina, Pythium insidiosum, Phytophthorainfestans, or plants such as soybean, oilseed rape, coconut, oil palm,safflower, flax, hemp, castor-oil plant, Calendula, peanut, cacao beanor sunflower, or yeasts such as Saccharomyces cerevisiae, with soybean,flax, oilseed rape, safflower, sunflower, Calendula, Mortierella orSaccharomyces cerevisiae being especially preferred. In principle, hostorganisms are, in addition to the abovementioned transgenic organisms,also transgenic animals, advantageously nonhuman animals, for example C.elegans. Further suitable host cells and organisms have already beendescribed extensively above.

Transgenic plants which comprise the polyunsaturated fatty acidssynthesized in the process according to the invention can advantageouslybe marketed directly without there being any need for the oils, lipidsor fatty acids synthesized to be isolated. Plants for the processaccording to the invention are listed as meaning intact plants and allplant parts, plant organs or plant parts such as leaf, stem, seeds,root, tubers, anthers, fibers, root hairs, stalks, embryos, calli,cotelydons, petioles, harvested material, plant tissue, reproductivetissue and cell cultures which are derived from the transgenic plantand/or can be used for bringing about the transgenic plant. In thiscontext, the seed comprises all parts of the seed such as the seedcoats, epidermal cells, seed cells, endosperm or embryonic tissue.However, the compounds produced in the process according to theinvention can also be isolated from the organisms, advantageouslyplants, in the form of their oils, fats, lipids and/or free fatty acids.Polyunsaturated fatty acids produced by this process can be obtained byharvesting the organisms, either from the crop in which they grow, orfrom the field. This can be done via pressing or extraction of the plantparts, preferably the plant seeds. In this context, the oils, fats,lipids and/or free fatty acids can be obtained by what is known ascold-beating or cold-pressing without applying heat. To allow forgreater ease of disruption of the plant parts, specifically the seeds,they are previously comminuted, steamed or roasted. The seeds which havebeen pretreated in this manner can subsequently be pressed or extractedwith solvent such as warm hexane. The solvent is subsequently removed.In the case of microorganisms, the latter are, after harvesting, forexample extracted directly without further processing steps or else,after disruption, extracted via various methods with which the skilledworker is familiar. In this manner, more than 96% of the compoundsproduced in the process can be isolated. Thereafter, the resultingproducts are processed further, i.e. refined. In this process, forexample the plant mucilages and suspended matter are first removed. Whatis known as desliming can be effected enzymatically or, for example,chemico-physically by addition of acid such as phosphoric acid.Thereafter, the free fatty acids are removed by treatment with a base,for example sodium hydroxide solution. The resulting product is washedthoroughly with water to remove the alkali remaining in the product andthen dried. To remove the pigments remaining in the product, theproducts are subjected to bleaching, for example using fuller's earth oractive charcoal. At the end, the product is deodorized, for exampleusing steam.

The PUFAs or LCPUFAs produced by this process are preferably C₁₈-, C₂₀-or C₂₂-fatty acid molecules, advantageously C₂₀- or C₂₂-fatty acidmolecules, with at least two double bonds in the fatty acid molecule,preferably three, four, five or six double bonds. These C₁₈-, C₂₀- orC₂₂-fatty acid molecules can be isolated from the organism in the formof an oil, a lipid or a free fatty acid. Suitable organisms are, forexample, those mentioned above. Preferred organisms are transgenicplants.

One embodiment of the invention is therefore oils, lipids or fatty acidsor fractions thereof which have been produced by the above-describedprocess, especially preferably oil, lipid or a fatty acid compositioncomprising PUFAs and being derived from transgenic plants.

As described above, these oils, lipids or fatty acids advantageouslycomprise 6 to 15% of palmitic acid, 1 to 6% of stearic acid, 7-85% ofoleic acid, 0.5 to 8% of vaccenic acid, 0.1 to 1% of arachic acid, 7 to25% of saturated fatty acids, 8 to 85% of monounsaturated fatty acidsand 60 to 85% of polyunsaturated fatty acids, in each case based on 100%and on the total fatty acid content of the organisms.

Advantageous polyunsaturated fatty acids which are present in the fattyacid esters or fatty acid mixtures are preferably at least 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1% of arachidonic acid, based onthe total fatty acid content. Moreover, the fatty acid esters or fattyacid mixtures which have been produced by the process of the inventionadvantageously comprise fatty acids selected from the group of the fattyacids erucic acid (13-docosaenoic acid), sterculic acid(9,10-methyleneoctadec-9-enoic acid), malvalic acid(8,9-methyleneheptadec-8-enoic acid), chaulmoogric acid(cyclopentenedodecanoic acid), furan fatty acid(9,12-epoxyoctadeca-9,11-dienoic acid), vernolic acid(9,10-epoxyoctadec-12-enoic acid), tariric acid (6-octadecynoic acid),6-nonadecynoic acid, santalbic acid (t11-octadecen-9-ynoic acid),6,9-octadecenynoic acid, pyrulic acid (t10-heptadecen-8-ynoic acid),crepenynic acid (9-octadecen-12-ynoic acid), 13,14-dihydrooropheic acid,octadecen-13-ene-9,11-diynoic acid, petroselenic acid(cis-6-octadecenoic acid), 9c,12t-octadecadienoic acid, calendulic acid(8t10t12c-octadecatrienoic acid), catalpic acid(9t11t13c-octadecatrienoic acid), eleostearic acid (9c1t13t-octadecatrienoic acid), jacaric acid (8c10t12c-octadecatrienoicacid), punicic acid (9c11t13c-octadecatrienoic acid), parinaric acid(9c11t13t15c-octadecatetraenoic acid), pinolenic acid(all-cis-5,9,12-octadecatrienoic acid), laballenic acid(5,6-octadecadienallenic acid), ricinoleic acid (12-hydroxyoleic acid)and/or coriolic acid (13-hydroxy-9c,11t-octadecadienoic acid).

The abovementioned fatty acids are, as a rule, advantageously only foundin traces in the fatty acid esters or fatty acid mixtures produced bythe process according to the invention, that is to say that, based onthe total fatty acids, they occur to less than 30%, preferably to lessthan 25%, 24%, 23%, 22% or 21%, especially preferably to less than 20%,15%, 10%, 9%, 8%, 7%, 6% or 5%, very especially preferably to less than4%, 3%, 2% or 1%. The fatty acid esters or fatty acid mixtures producedby the process according to the invention advantageously comprise lessthan 0.1%, based on the total fatty acids, or no butyric acid, nocholesterol, no clupanodonic acid (=docosapentaenoic acid,C22:5^(Δ4,8,12,15,21)) and no nisinic acid (tetracosahexaenoic acid,C23:6^(Δ3,8,12,15,18,21)).

The oils, lipids or fatty acids according to the invention preferablycomprise at least 0.5%, 1%, 2%, 3%, 4% or 5%, advantageously at least6%, 7%, 8%, 9% or 10%, especially advantageously at least 11%, 12%, 13%,14% or 15% of ARA or at least 0.5%, 1%, 2%, 3%, 4% or 5%, advantageouslyat least 6% or 7%, especially advantageously at least 8%, 9% or 10% ofEPA and/or DHA, based on the total fatty acid content of the productionorganism, advantageously of a plant, especially advantageously of an oilcrop plant such as soybean, oilseed rape, coconut, oil palm, safflower,flax, hemp, castor-oil plant, Calendula, peanut, cacao bean, sunflower,or the abovementioned further mono- or dicotyledonous oil crop plants.

A further embodiment according to the invention is the use of the oil,lipid, the fatty acids and/or the fatty acid composition in feedstuffs,foodstuffs, cosmetics or pharmaceuticals. The oils, lipids, fatty acidsor fatty acid mixtures according to the invention can be used in themanner with which the skilled worker is familiar for mixing with otheroils, lipids, fatty acids or fatty acid mixtures of animal origin, suchas, for example, fish oils. These oils, lipids, fatty acids or fattyacid mixtures, which are composed of vegetable and animal constituents,may also be used for the preparation of feedstuffs, foodstuffs,cosmetics or pharmaceuticals.

The term “oil”, “lipid” or “fat” is understood as meaning a fatty acidmixture comprising unsaturated, saturated, preferably esterified, fattyacid(s). The oil, lipid or fat is preferably high in polyunsaturatedfree or, advantageously, esterified fatty acid(s), in particularlinoleic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonicacid, α-linolenic acid, stearidonic acid, eicosatetraenoic acid,eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid.The amount of unsaturated esterified fatty acids preferably amounts toapproximately 30%, a content of 50% is more preferred, a content of 60%,70%, 80% or more is even more preferred. For the analysis, the fattyacid content can, for example, be determined by gas chromatography afterconverting the fatty acids into the methyl esters bytransesterification. The oil, lipid or fat can comprise various othersaturated or unsaturated fatty acids, for example calendulic acid,palmitic acid, palmitoleic acid, stearic acid, oleic acid and the like.The content of the various fatty acids in the oil or fat can vary, inparticular depending on the starting organism.

The polyunsaturated fatty acids with advantageously at least two doublebonds which are produced in the process are, as described above, forexample sphingolipids, phosphoglycerides, lipids, glycolipids,phospholipids, monoacylglycerol, diacylglycerol, triacylglycerol orother fatty acid esters.

Starting from the polyunsaturated fatty acids with advantageously atleast five or six double bonds, which acids have been prepared in theprocess according to the invention, the polyunsaturated fatty acidswhich are present can be liberated for example via treatment withalkali, for example aqueous KOH or NaOH, or acid hydrolysis,advantageously in the presence of an alcohol such as methanol orethanol, or via enzymatic cleavage, and isolated via, for example, phaseseparation and subsequent acidification via, for example, H₂SO₄. Thefatty acids can also be liberated directly without the above-describedprocessing step.

After their introduction into an organism, advantageously a plant cellor plant, the nucleic acids used in the process can either be present ona separate plasmid or, advantageously, integrated into the genome of thehost cell. In the case of integration into the genome, integration canbe random or else be effected by recombination such that the native geneis replaced by the copy introduced, whereby the production of thedesired compound by the cell is modulated, or by the use of a gene in“trans”, so that the gene is operably linked with a functionalexpression unit which comprises at least one sequence which ensures theexpression of a gene and at least one sequence which ensures thepolyadenylation of a functionally transcribed gene. The nucleic acidsare advantageously introduced into the organisms via multiexpressioncassettes or constructs for multiparallel expression, advantageouslyinto the plants for the multiparallel seed-specific expression of genes.

Mosses and algae are the only known plant systems which producesubstantial amounts of polyunsaturated fatty acids such as arachidonicacid (ARA) and/or eicosapentaenoic acid (EPA) and/or docosahexaenoicacid (DHA). Mosses comprise PUFAs in membrane lipids, while algae,organisms which are related to algae and a few fungi also accumulatesubstantial amounts of PUFAs in the triacylglycerol fraction. This iswhy nucleic acid molecules which are isolated from such strains thatalso accumulate PUFAs in the triacylglycerol fraction are particularlyadvantageous for the process according to the invention and thus for themodification of the lipid and PUFA production system in a host, inparticular plants such as oil crops, for example oilseed rape, canola,linseed, hemp, soybeans, sunflowers and borage. They can therefore beused advantageously in the process according to the invention.

Substrates which are suitable for the polypeptides according to theinvention or of the polypeptide of the fatty acid or lipid metabolismselected from the group acyl-CoA dehydrogenase(s), acyl-ACP [=acylcarrier protein]desaturase(s), acyl-ACP thioesterase(s), fatty acidacyltransferase(s), acyl-CoA: lysophospholipid acyltransferase(s), fattyacid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme Acarboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s),fatty acid acetylenase(s), lipoxygenase(s), triacylglycerol lipase(s),allene oxide synthase(s), hydroperoxide lyase(s) or fatty acidelongase(s) are advantageously C₁₆-, C₁₈- or C₂₀-fatty acids. The fattyacids converted as substrates in the process are preferably converted inthe form of their acyl-CoA esters and/or their phospholipid esters.

To produce the long-chain PUFAs according to the invention, thepolyunsaturated C₁₈-fatty acids must first be desaturated by theenzymatic activity of a desaturase and subsequently be elongated by atleast two carbon atoms via an elongase. After one elongation cycle, thisenzyme activity gives C₂₀-fatty acids and after two elongation cyclesC₂₂-fatty acids. The activity of the desaturases and elongases used inthe process according to the invention preferably leads to C₁₈-, C₂₀-and/or C₂₂-fatty acids, advantageously with at least two double bonds inthe fatty acid molecule, preferably with three, four, five or six doublebonds, especially preferably to give C₂₀- and/or C₂₂-fatty acids with atleast two double bonds in the fatty acid molecule, preferably withthree, four, five or six double bonds, very especially preferably withfive or six double bonds in the molecule. After a first desaturation andthe elongation have taken place, further desaturation and elongationsteps such as, for example, such a desaturation in the Δ5 and Δ4position may take place. Products of the process according to theinvention which are especially preferred are dihomo-γ-linolenic acid,arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid and/ordocosahexaenoic acid. The C₂₀-fatty acids with at least two double bondsin the fatty acid can be elongated by the enzymatic activity accordingto the invention in the form of the free fatty acid or in the form ofthe esters, such as phospholipids, glycolipids, sphingolipids,phosphoglycerides, monoacylglycerol, diacylglycerol or triacylglycerol.

The preferred biosynthesis site of the fatty acids, oils, lipids or fatsin the plants which are advantageously used is, for example, in generalthe seed or cell strata of the seed, so that seed-specific expression ofthe nucleic acids used in the process is sensible. However, it isobvious that the biosynthesis of fatty acids, oils or lipids need not belimited to the seed tissue, but can also take place in a tissue-specificmanner in all the other parts of the plant, for example in epidermalcells or in the tubers.

If microorganism such as yeasts, such as Saccharomyces orSchizosaccharomyces, fungi such as Mortierella, Aspergillus,Phytophthora, Entomophthora, Mucor or Thraustochytrium, algae such asIsochrysis, Mantoniella, Ostreococcus, Phaeodactylum or Crypthecodiniumare used as organisms in the process according to the invention, theseorganisms are advantageously grown in fermentation cultures. Owing tothe use of the nucleic acids according to the invention which code for aΔ5-elongase, the polyunsaturated fatty acids produced in the process canbe increased by at least 5%, preferably by at least 10%, especiallypreferably by at least 20%, very especially preferably by at least 50%in comparison with the wild types of the organisms which do not comprisethe nucleic acids recombinantly.

In principle, the polyunsaturated fatty acids produced by the processaccording to the invention in the organisms used in the process can beincreased in two different ways. Advantageously, the pool of freepolyunsaturated fatty acids and/or the content of the esterifiedpolyunsaturated fatty acids produced via the process can be enlarged.Advantageously, the pool of esterified polyunsaturated fatty acids inthe transgenic organisms is enlarged by the process according to theinvention.

If microorganisms are used as organisms in the process according to theinvention, they are grown or cultured in the manner with which theskilled worker is familiar, depending on the host organism. As a rule,microorganisms are grown in a liquid medium comprising a carbon source,usually in the form of sugars, a nitrogen source, usually in the form oforganic nitrogen sources such as yeast extract or salts such as ammoniumsulfate, trace elements such as salts of iron, manganese and magnesiumand, if appropriate, vitamins, at temperatures of between 0° C. and 100°C., preferably between 10° C. and 60° C., while introducing oxygen gas.The pH of the nutrient liquid can either be kept constant, that is tosay regulated during the culturing period, or not. The cultures can begrown batchwise, semi-batchwise or continuously. Nutrients can beprovided at the beginning of the fermentation or fed in semicontinuouslyor continuously. The polyunsaturated fatty acids produced can beisolated from the organisms as described above by processes known to theskilled worker, for example by extraction, distillation,crystallization, if appropriate precipitation with salt, and/orchromatography. To this end, the organisms can advantageously bedisrupted beforehand.

If the host organisms are microorganisms, the process according to theinvention is advantageously carried out at a temperature of between 0°C. and 95° C., preferably between 10° C. and 85° C., especiallypreferably between 15° C. and 75° C., very especially preferably between15° C. and 45° C.

In this process, the pH value is advantageously kept between pH 4 and12, preferably between pH 6 and 9, especially preferably between pH 7and 8.

The process according to the invention can be operated batchwise,semibatchwise or continuously. An overview over known cultivationmethods can be found in the textbook by Chmiel (Bioprozelltechnik 1.Einfuhrung in die Bioverfahrenstechnik [Bioprocess technology 1.Introduction to bioprocess technology](Gustav Fischer Verlag, Stuttgart,1991)) or in the textbook by Storhas (Bioreaktoren und periphereEinrichtungen [Bioreactors and peripheral equipment](Vieweg Verlag,Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must suitably meet the requirements of thestrains in question. Descriptions of culture media for variousmicroorganisms can be found in the textbook “Manual of Methods forGeneral Bacteriology” of the American Society for Bacteriology(Washington D. C., USA, 1981).

As described above, these media which can be employed in accordance withthe invention usually comprise one or more carbon sources, nitrogensources, inorganic salts, vitamins and/or trace elements.

Preferred carbon sources are sugars, such as mono-, di- orpolysaccharides. Examples of very good carbon sources are glucose,fructose, mannose, galactose, ribose, sorbose, ribulose, lactose,maltose, sucrose, raffinose, starch or cellulose. Sugars can also beadded to the media via complex compounds such as molasses or otherby-products from sugar raffination. The addition of mixtures of avariety of carbon sources may also be advantageous. Other possiblecarbon sources are oils and fats such as, for example, soya oil,sunflower oil, peanut oil and/or coconut fat, fatty acids such as, forexample, palmitic acid, stearic acid and/or linoleic acid, alcoholsand/or polyalcohols such as, for example, glycerol, methanol and/orethanol, and/or organic acids such as, for example, acetic acid and/orlactic acid.

Nitrogen sources are usually organic or inorganic nitrogen compounds ormaterials comprising these compounds. Examples of nitrogen sourcescomprise ammonia in liquid or gaseous form or ammonium salts such asammonium sulfate, ammonium chloride, ammonium phosphate, ammoniumcarbonate or ammonium nitrate, nitrates, urea, amino acids or complexnitrogen sources such as cornsteep liquor, soya meal, soya protein,yeast extract, meat extract and others. The nitrogen sources can be usedindividually or as a mixture.

Inorganic salt compounds which may be present in the media comprise thechloride, phosphorus and sulfate salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

Inorganic sulfur-containing compounds such as, for example, sulfates,sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or elseorganic sulfur compounds such as mercaptans and thiols may be used assources of sulfur for the production of sulfur-containing finechemicals, in particular of methionine.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used assources of phosphorus.

Chelating agents may be added to the medium in order to keep the metalions in solution. Particularly suitable chelating agents includedihydroxyphenols such as catechol or protocatechuate and organic acidssuch as citric acid.

The fermentation media used according to the invention for culturingmicroorganisms usually also comprise other growth factors such asvitamins or growth promoters, which include, for example, biotin,riboflavin, thiamine, folic acid, nicotinic acid, panthothenate andpyridoxine. Growth factors and salts are frequently derived from complexmedia components such as yeast extract, molasses, cornsteep liquor andthe like. It is moreover possible to add suitable precursors to theculture medium. The exact composition of the media compounds heavilydepends on the particular experiment and is decided upon individuallyfor each specific case. Information on the optimization of media can befound in the textbook “Applied Microbiol. Physiology, A PracticalApproach” (Editors P. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp.53-73, ISBN 0 19 963577 3). Growth media can also be obtained fromcommercial suppliers, for example Standard 1 (Merck) or BHI (brain heartinfusion, DIFCO) and the like.

All media components are sterilized, either by heat (20 min at 1.5 barand 121° C.) or by filter sterilization. The components may besterilized either together or, if required, separately. All mediacomponents may be present at the start of the cultivation or addedcontinuously or batchwise, as desired.

The culture temperature is normally between 15° C. and 45° C.,preferably at from 25° C. to 40° C., and may be kept constant or may bealtered during the experiment. The pH of the medium should be in therange from 5 to 8.5, preferably around 7.0. The pH for cultivation canbe controlled during cultivation by adding basic compounds such assodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia oracidic compounds such as phosphoric acid or sulfuric acid. Foaming canbe controlled by employing antifoams such as, for example, fatty acidpolyglycol esters. To maintain the stability of plasmids it is possibleto add to the medium suitable substances having a selective effect, forexample antibiotics. Aerobic conditions are maintained by introducingoxygen or oxygen-containing gas mixtures such as, for example, ambientair, into the culture. The temperature of the culture is normally 20° C.to 45° C. and preferably 25° C. to 40° C. The culture is continued untilformation of the desired product is at a maximum. This aim is normallyachieved within 10 to 160 hours.

The fermentation broths obtained in this way, in particular thosecontaining polyunsaturated fatty acids, usually contain a dry mass offrom 7.5 to 25% by weight.

The fermentation broth can then be processed further. The biomass may,according to requirement, be removed completely or partially from thefermentation broth by separation methods such as, for example,centrifugation, filtration, decanting or a combination of these methodsor be left completely in said broth. It is advantageous to process thebiomass after its separation.

However, the fermentation broth can also be thickened or concentratedwithout separating the cells, using known methods such as, for example,with the aid of a rotary evaporator, thin-film evaporator, falling-filmevaporator, by reverse osmosis or by nanofiltration. Finally, thisconcentrated fermentation broth can be processed to obtain the fattyacids present therein.

The polynucleotides or polypeptides of the present invention which areinvolved in the metabolism of lipids and fatty acids, PUFA cofactors andenzymes or in the transport of lipophilic compounds across membranes areused in the process according to the invention for the modulation of theproduction of PUFAs in transgenic organisms, advantageously in plants,such as maize, wheat, rye, oats, triticale, rice, barley, soybean,peanut, cotton, Linum species such as linseed or flax, Brassica speciessuch as oilseed rape, canola and turnip rape, pepper, sunflower, borage,evening primrose and Tagetes, Solanaceae plants such as potato, tobacco,eggplant and tomato, Vicia species, pea, cassava, alfalfa, bushy plants(coffee, cacao, tea), Salix species, trees (oil palm, coconut) andperennial grasses and fodder crops, either directly (for example whenthe overexpression or optimization of a fatty acid biosynthesis proteinhas a direct effect on the yield, production and/or productionefficiency of the fatty acid from modified organisms) and/or can have anindirect effect which nevertheless leads to an enhanced yield,production and/or production efficiency of the PUFAs or a reduction ofundesired compounds (for example when the modulation of the metabolismof lipids and fatty acids, cofactors and enzymes lead to modificationsof the yield, production and/or production efficiency or the compositionof the desired compounds within the cells, which, in turn, can affectthe production of one or more fatty acids).

The combination of various precursor molecules and biosynthesis enzymesleads to the production of various fatty acid molecules, which has adecisive effect on lipid composition, since polyunsaturated fatty acids(=PUFAs) are not only incorporated into triacylglycerol but also intomembrane lipids.

Brassicaceae, Boraginaceae, Primulaceae, or Linaceae are particularlysuitable for the production of PUFAs, for example stearidonic acid,eicosapentaenoic acid and docosahexaenoic acid. Linseed (Linumusitatissimum) is especially advantageously suitable for the productionof PUFAs with the nucleic acid sequences according to the invention,advantageously, as described, in combination with further desaturasesand elongases.

Lipid synthesis can be divided into two sections: the synthesis of fattyacids and their binding to sn-glycerol-3-phosphate, and the addition ormodification of a polar head group. Usual lipids which are used inmembranes comprise phospholipids, glycolipids, sphingolipids andphosphoglycerides. Fatty acid synthesis starts with the conversion ofacetyl-CoA into malonyl-CoA by acetyl-CoA carboxylase or into acetyl-ACPby acetyl transacylase. After a condensation reaction, these two productmolecules together form acetoacetyl-ACP, which is converted via a seriesof condensation, reduction and dehydratation reactions so that asaturated fatty acid molecule with the desired chain length is obtained.The production of the unsaturated fatty acids from these molecules iscatalyzed by specific desaturases, either aerobically by means ofmolecular oxygen or anaerobically (regarding the fatty acid synthesis inmicroorganisms, see F. C. Neidhardt et al. (1996) E. coli andSalmonella. ASM Press: Washington, D.C., pp. 612-636 and referencescited therein; Lengeler et al. (Ed.) (1999) Biology of Procaryotes.Thieme: Stuttgart, N.Y., and the references therein, and Magnuson, K.,et al. (1993) Microbiological Reviews 57:522-542 and the referencestherein). To undergo the further elongation steps, the resultingphospholipid-bound fatty acids must be returned to the fatty acid CoAester pool from the phospholipids. This is made possible byacyl-CoA:lysophospholipid acyltransferases. Moreover, these enzymes arecapable of transferring the elongated fatty acids from the CoA estersback to the phospholipids. If appropriate, this reaction sequence can befollowed repeatedly.

Examples of precursors for the biosynthesis of PUFAs are oleic acid,linoleic acid and linolenic acid. These C₁₈-carbon fatty acids must beelongated to C₂₀ and C₂₂ in order to obtain fatty acids of the eicosaand docosa chain type. With the aid of the desaturases used in theprocess, such as the Δ12-, Δ4-, Δ5- and Δ6-desaturases and/or Δ5-,Δ6-elongases, arachidonic acid, eicosapentaenoic acid, docosapentaenoicacid or docosahexaenoic acid, advantageously eicosapentaenoic acidand/or docosahexaenoic acid, can be produced and subsequently employedin various applications regarding foodstuffs, feedstuffs, cosmetics orpharmaceuticals. C₂₀- and/or C₂₂-fatty acids with at least two,advantageously at least three, four, five or six, double bonds in thefatty acid molecule, preferably C₂₀- or C₂₂-fatty acids withadvantageously four, five or six double bonds in the fatty acidmolecule, can be prepared using the abovementioned enzymes. Desaturationmay take place before or after elongation of the fatty acid in question.This is why the products of the desaturase activities and the furtherdesaturation and elongation steps which are possible result in preferredPUFAs with a higher degree of desaturation, including a furtherelongation from C₂₀- to C₂₂-fatty acids, to fatty acids such asγ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, stearidonicacid, eicosatetraenoic acid or eicosapentaenoic acid. Substrates of thedesaturases and elongases used in the process according to the inventionare C₁₆-, C₁₈- or C₂₀-fatty acids such as, for example, linoleic acid,γ-linolenic acid, α-linolenic acid, dihomo-γ-linolenic acid,eicosatetraenoic acid or stearidonic acid. Preferred substrates arelinoleic acid, γ-linolenic acid and/or α-linolenic acid,dihomo-γ-linolenic acid or arachidonic acid, eicosatetraenoic acid oreicosapentaenoic acid. The synthesized C₂₀- or C₂₂-fatty acids with atleast two, three, four, five or six double bonds in the fatty acid areobtained in the process according to the invention in the form of thefree fatty acid or in the form of their esters, for example in the formof their glycerides.

The term “glyceride” is understood as meaning a glycerol esterified withone, two or three carboxyl radicals (mono-, di- or triglyceride).“Glyceride” is also understood as meaning a mixture of variousglycerides. The glyceride or glyceride mixture may comprise furtheradditions, for example free fatty acids, antioxidants, proteins,carbohydrates, vitamins and/or other substances.

For the purposes of the invention, a “glyceride” is furthermoreunderstood as meaning glycerol derivatives. In addition to theabove-described fatty acid glycerides, these also includeglycerophospholipids and glyceroglycolipids. Preferred examples whichmay be mentioned in this context are the glycerophospholipids such aslecithin (phosphatidylcholine), cardiolipin, phosphatidylglycerol,phosphatidylserine and alkylacylglycerophospholipids.

Furthermore, fatty acids must subsequently be translocated to variousmodification sites and incorporated into the triacylglycerol storagelipid. A further important step in lipid synthesis is the transfer offatty acids to the polar head groups, for example by glycerol fatty acidacyltransferase (see Frentzen, 1998, Lipid, 100(4-5):161-166).

Publications on plant fatty acid biosynthesis and on the desaturation,the lipid metabolism and the membrane transport of lipidic compounds, onbeta-oxidation, fatty acid modification and cofactors, triacylglycerolstorage and triacylglycerol assembly, including the references therein,see the following papers: Kinney, 1997, Genetic Engineering, Ed.: JKSetlow, 19:149-166; Ohlrogge and Browse, 1995, Plant Cell 7:957-970;Shanklin and Cahoon, 1998, Annu. Rev. Plant Physiol. Plant Mol. Biol.49:611-641; Voelker, 1996, Genetic Engineering, Ed.: JK Setlow,18:111-13; Gerhardt, 1992, Prog. Lipid R. 31:397-417; Gihnemann-Schafer& Kindl, 1995, Biochim. Biophys Acta 1256:181-186; Kunau et al., 1995,Prog. Lipid Res. 34:267-342; Stymne et al., 1993, in: Biochemistry andMolecular Biology of Membrane and Storage Lipids of Plants, Ed.: Murataand Somerville, Rockville, American Society of Plant Physiologists,150-158, Murphy & Ross 1998, Plant Journal. 13(1):1-16.

The PUFAs produced in the process comprise a group of molecules whichhigher animals are no longer capable of synthesizing and must thereforetake up, or which higher animals are no longer capable of synthesizingthemselves in sufficient quantity and must therefore take up additionalquantities, although they can be synthesized readily by other organismssuch as bacteria; for example, cats are no longer capable ofsynthesizing arachidonic acid.

Phospholipids for the purposes of the invention are understood asmeaning phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidyl-glycerol and/or phosphatidylinositol,advantageously phosphatidylcholine. The terms production or productivityare known in the art and encompass the concentration of the fermentationproduct (compounds of the formula I) which is formed within a specificperiod of time and in a specific fermentation volume (for example kg ofproduct per hour per liter). It also comprises the productivity within aplant cell or a plant, that is to say the content of the desired fattyacids produced in the process relative to the content of all fatty acidsin this cell or plant. The term production efficiency comprises the timerequired for obtaining a specific production quantity (for example thetime required by the cell to establish a certain throughput rate of afine chemical). The term yield or product/carbon yield is known in theart and comprises the efficiency of the conversion of the carbon sourceinto the product (i.e. the fine chemical). This is usually expressed forexample as kg of product per kg of carbon source. By increasing theyield or production of the compound, the amount of the moleculesobtained of this compound, or of the suitable molecules of this compoundobtained, in a specific culture quantity over a specified period of timeis increased. The terms biosynthesis or biosynthetic pathway are knownin the art and comprise the synthesis of a compound, preferably anorganic compound, by a cell from intermediates, for example in amulti-step and strongly regulated process. The terms catabolism orcatabolic pathway are known in the art and comprise the cleavage of acompound, preferably of an organic compound, by a cell to givecatabolites (in more general terms, smaller or less complex molecules),for example in a multi-step and strongly regulated process. The termmetabolism is known in the art and comprises the totality of thebiochemical reactions which take place in an organism. The metabolism ofa certain compound (for example the metabolism of a fatty acid) thuscomprises the totality of the biosynthetic pathways, modificationpathways and catabolic pathways of this compound in the cell whichrelate to this compound.

By employing, in the process according to the invention, thepolynucleotides according to the invention and optionally furtherpolynucleotides which code for enzymes of the lipid or fatty acidmetabolism it is possible to achieve various advantageous effects.

Thus, it is possible to influence the yield, production and/orproduction efficiency of the polyunsaturated fatty acids in a plant,preferably in an oil crop plant, or in a microorganism. The number oractivity of the polypeptides or polynucleotides according to theinvention can be increased, so that larger amounts of the gene productsand, ultimately, larger amounts of the compounds of the general formulaI are produced. A de novo synthesis in an organism, which, before thegene(s) in question has/have been introduced, had been lacking theactivity and capability of biosynthesizing the compounds, is alsopossible. The same applies analogously to the combination with furtherdesaturases or elongases or further enzymes of the fatty acid and lipidmetabolism. The use of a variety of divergent sequences, i.e. sequenceswhich differ at the DNA sequence level, may be advantageous in thiscontext, or else the use of gene expression promoters which makespossible a different gene expression as far as timing is concerned, forexample as a function of the degree of maturity of a seed or oil-storingtissue.

By introducing, into an organism, a polynucleotide according to theinvention alone or in combination with other genes into a cell it ispossible not only to increase the biosynthetic flow towards the endproduct, but also to increase, or to create de novo, the correspondingtriacylglycerol composition. Equally, the number or activity of othergenes which are required for the import of nutrients for thebiosynthesis of one or more fatty acids, oils, polar and/or neutrallipids, so that the concentration of these precursors, cofactors orintermediates within the cells or within the storage compartment isincreased, whereby the ability of the cells to produce PUFAs is furtherenhanced. By optimizing the activity, or increasing the number, of oneor more polynucleotides or polypeptides according to the invention whichare involved in the biosynthesis of these compounds, or by destroyingthe activity of one or more genes which are involved in the degradationof these compounds, it may be possible to increase the yield, productionand/or production efficiency of fatty acid and lipid molecules fromorganisms, in particular from plants. The fatty acids obtained in theprocess are suitable as starting materials for the chemical synthesis offurther products of interest. For example, they can be used for thepreparation of pharmaceuticals, foodstuffs, animal feeds or cosmetics,either alone or in combination with one another.

It can be seen from what has been said above that the invention alsorelates to a process for the preparation of an oil, lipid or fatty acidcomposition, comprising the steps of the process according to theinvention and the further step of formulating the substance as an oil,lipid or fatty acid composition.

In a preferred embodiment of this process, the oil, lipid or fatty acidcomposition is formulated further to give a pharmaceutical, a cosmeticproduct, a foodstuff, a feeding stuff, preferably fish food, or a foodsupplement.

Finally, the invention relates to the principle of using thepolynucleotide, the vector, the host cell, the polypeptide or thetransgenic, nonhuman organism of the present invention for theproduction of an oil, lipid or fatty acid composition. The latter isthen preferably to be employed as pharmaceutical, cosmetic product,foodstuff, feeding stuff, preferably fish food, or food supplement.

The content of all the references, patent applications, patents andpublished patent applications cited in the present patent application isherewirh incorporated by reference to the respective specificdisclosure.

FIGURES

FIG. 1 shows a sequence alignment of the Δ5- and Δ6-elongase amino acidsequences from O. lucimarinus (d5-elo-Olu (SEQ ID NO: 12); d6-elo-Olu(SEQ ID NO: 16)), O. tauri (d5-elo-Ota (SEQ ID NO: 18); d6-elo-Ota (SEQID NO: 20)), and T. pseudonana (d5-elo-Tps (SEQ ID NO: 34); d6-elo-Tps(SEQ ID NO: 36)) in the ClustalW comparison.

FIG. 2 shows a sequence alignment of the Δ4-desaturase amino acidsequences from O. lucimarinus (d4-des-Olu (SEQ ID NO: 6)), O. tauri(d4-des-Ota (SEQ ID NO: 24)), and T. pseudonana (d4-des-Tps (SEQ ID NO:42)) in the ClustalW comparison.

FIG. 3 shows a sequence alignment of the Δ5-desaturase amino acidsequences from O. lucimarinus (first d5-des-Olu (SEQ ID NO: 8); secondd5-des-Olu (SEQ ID NO: 10)), O. tauri (d5-des-Ota (SEQ ID NO: 28)), andT. pseudonana (d5-des-Tps (SEQ ID NO: 40)) in the ClustalW comparison.

FIG. 4 shows a sequence alignment of the Δ6-desaturase amino acidsequences from O. lucimarinus (d6-des-Olu (SEQ ID NO: 14)), O. tauri(d6-des-Ota (SEQ ID NO: 30)), and T. pseudonana (d6-des-Tps (SEQ ID NO:38)) in the ClustalW comparison.

FIG. 5 shows a sequence alignment of the Δ12-desaturase amino acidsequences from O. lucimarinus (first d12-des-OI (SEQ ID NO: 4); secondd12-des-OI (SEQ ID NO: 2)), O. tauri (d12-des-Ot (SEQ ID NO: 32)), andT. pseudonana (d12-des-Tp (SEQ ID NO: 44)) in the ClustalW comparison.

FIG. 6 shows the gas-chromatographic determination of the fatty acidsfrom yeasts which have been transformed with the plasmid pYES (A, B) orpYES-D5EIo(OI) (C). The fatty acid 20:4Δ5,8,11,14 was fed (B, C).

FIG. 7 shows the gas-chromatographic determination of the fatty acidsfrom yeasts which have been transformed with the plasmid pYES (A, B, C)or pYES-D6EIo(OI) (D,E). The fatty acids 18:3Δ6,9,12 or 18:4Δ6,9,12,15were fed (B, D) and (C, E), respectively.

FIG. 8 shows the gas-chromatographic determination of the fatty acidsfrom yeasts which have been transformed with the plasmid pYES (A, B) orpYES-D5Des(OI_2) (C). The fatty acid 20:3Δ5,8,11,14 was fed (B) and (C).

FIG. 9 shows the gas-chromatographic determination of the fatty acidsfrom yeasts which have been transformed with the plasmid pYES (A) orpYES-D12Des(OI) (B).

FIG. 10 shows the gas-chromatographic determination of the fatty acidsfrom yeast. pYes-d5Des(OI_1) in yeast strain InvSc without addition offatty acids (A); pYes-d5Des(OI_1) in yeast strain InvSc after additionof the fatty acid 20:3n-6 (B), pYes-d5Des(OI_1) in yeast strain InvScafter addition of the fatty acid 20:4n-3 (C).

The present invention is illustrated in greater detail by the exampleswhich follow, which are not to be construed as limiting.

EXAMPLES Example 1 General Cloning Methods

The cloning methods such as, for example, restriction cleavages, agarosegel electrophoresis, purification of DNA fragments, transfer of nucleicacids to nitrocellulose and nylon membranes, linkage of DNA fragments,transformation of Escherichia coli cells, bacterial cultures and thesequence analysis of recombinant DNA were carried out as described bySambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN0-87969-309-6).

Example 2 Sequence Analysis of Recombinant DNA

Recombinant DNA molecules were sequenced with an ABI laser fluorescenceDNA sequencer by the method of Sanger (Sanger et al. (1977) Proc. Natl.Acad. Sci. USA74, 5463-5467). Fragments obtained by polymerase chainreaction were sequenced and verified to avoid polymerase errors inconstructs to be expressed.

Example 3 Lipid Extraction from Yeasts

The effect of the genetic modification in plants, fungi, algae, ciliatesor on the production of a desired compound (such as a fatty acid) can bedetermined by growing the modified microorganisms or the modified plantunder suitable conditions (such as those described above) and analyzingthe medium and/or the cellular components for the elevated production ofthe desired product (i.e. of the lipids or a fatty acid). Theseanalytical techniques are known to the skilled worker and comprisespectroscopy, thin-layer chromatography, various types of stainingmethods, enzymatic and microbiological methods and analyticalchromatography such as high-performance liquid chromatography (see, forexample, Ullman, Encyclopedia of Industrial Chemistry, Vol. Δ2, p. 89-90and p. 443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987)“Applications of HPLC in Biochemistry” in: Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993)Biotechnology, Vol. 3, Chapter IIl: “Product recovery and purification”,p. 469-714, VCH: Weinheim; Belter, P. A., et al. (1988) Bioseparations:downstream processing for Biotechnology, John Wiley and Sons; Kennedy,J. F., and Cabral, J. M. S. (1992) Recovery processes for biologicalMaterials, John Wiley and Sons; Shaeiwitz, J. A., and Henry, J. D.(1988) Biochemical Separations, in: Ullmann's Encyclopedia of IndustrialChemistry, Vol. B3; Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, NoyesPublications).

In addition to the abovementioned processes, plant lipids are extractedfrom plant material as described by Cahoon et al. (1999) Proc. Natl.Acad. Sci. USA 96 (22):12935-12940 and Browse et al. (1986) AnalyticBiochemistry 152:141-145. The qualitative and quantitative analysis oflipids or fatty acids is described by Christie, William W., Advances inLipid Methodology, Ayr/Scotland: Oily Press (Oily Press Lipid Library;2); Christie, William W., Gas Chromatography and Lipids. A PracticalGuide-Ayr, Scotland: Oily Press, 1989, Repr. 1992, IX, 307 pp. (OilyPress Lipid Library; 1); “Progress in Lipid Research”, Oxford: PergamonPress, 1 (1952)-16 (1977) under the title: Progress in the Chemistry ofFats and Other Lipids CODEN.

In addition to measuring the end product of the fermentation, it is alsopossible to analyze other components of the metabolic pathways which areused for the production of the desired compound, such as intermediatesand by-products, in order to determine the overall production efficiencyof the compound. The analytical methods comprise measuring the amount ofnutrients in the medium (for example sugars, hydrocarbons, nitrogensources, phosphate and other ions), measuring the biomass compositionand the growth, analyzing the production of conventional metabolites ofbiosynthetic pathways and measuring gases which are generated during thefermentation. Standard methods for these measurements are described inApplied Microbial Physiology; A Practical Approach, P. M. Rhodes and P.F. Stanbury, Ed., IRL Press, p. 103-129; 131-163 and 165-192 (ISBN:0199635773) and references cited therein.

One example is the analysis of fatty acids (abbreviations: FAME, fattyacid methyl ester; GC-MS, gas liquid chromatography/mass spectrometry;TAG, triacylglycerol; TLC, thin-layer chromatography).

The unambiguous proof for the presence of fatty acid products can beobtained by analyzing recombinant organisms using standard analyticalmethods: GC, GC-MS or TLC, as described on several occasions by Christieand the references therein (1997, in: Advances on Lipid Methodology,Fourth Edition: Christie, Oily Press, Dundee, 119-169; 1998,Gaschromatographie-Massenspektrometrie-Verfahren [Gaschromatography/mass spectrometry methods], Lipide 33:343-353).

The material to be analyzed can be disrupted by sonication, grinding ina glass mill, liquid nitrogen and grinding or via other applicablemethods. After disruption, the material must be centrifuged. Thesediment is resuspended in distilled water, heated for 10 minutes at100° C., cooled on ice and recentrifuged, followed by extraction for onehour at 90° C. in 0.5 M sulfuric acid in methanol with 2%dimethoxypropane, which leads to hydrolyzed oil and lipid compounds,which give transmethylated lipids. These fatty acid methyl esters areextracted in petroleum ether and finally subjected to a GC analysisusing a capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25μm, 0.32 mm) at a temperature gradient of between 170° C. and 240° C.for 20 minutes and 5 minutes at 240° C. The identity of the resultingfatty acid methyl esters must be defined using standards which areavailable from commercial sources (i.e. Sigma).

Example 4 Cloning and Characterization of Elongase Genes fromOstreococcus lucimarinus

By searching for conserved regions in the protein sequences in elongasegenes, it was possible to identify two sequences with correspondingmotifs in an Ostreococcus lucimarinus sequence database. In a furtherstep, the genes were characterized by means of sequence alignment, geneprediction and the search for coding regions. The following codingregions were found:

TABLE 1 Coding regions Name of gene SEQ ID Amino acids D5Elo(OI) 12 298D6Elo(OI) 16 287

FIG. 1 shows the sequence similarities with other algae (Ostreococcustauri, Thalassiosira pseudonana) for the various elongase amino acidsequences in the ClustalW sequence alignment. Surprisingly, the O.lucimarinus sequences differ markedly from the other algae in theiramino acid sequence.

TABLE 2 Sequence identities of individual elongases Name of gene SEQ IDOrganism Identity in % D5Elo(OI) 28 O. lucimarinus 100 O. tauri 77 T.pseudonana 21 D6Elo(OI) 32 O. lucimarinus 100 O. tauri 71 T. pseudonana25

The cloning procedure was carried out as follows:

40 ml of an Ostreococcus lucimarinus culture in the stationary phasewere spun down and resuspended in 100 μl of double-distilled water andstored at −20° C. The relevant genomic DNAs were amplified based on thePCR method. The corresponding primer pairs were selected in such a waythat they contained the yeast consensus sequence for highly efficienttranslation (Kozak, Cell 1986, 44:283-292) next to the start codon. Theamplification of the DNAs was carried out using in each case 1 μl ofdefrosted cells, 200 μM dNTPs, 2.5 U Taq polymerase and 100 pmol of eachprimer in a total volume of 50 μl. The conditions for the PCR were asfollows: first denaturation at 95° C. for 5 minutes, followed by 30cycles at 94° C. for 30 seconds, 55° C. for 1 minute and 72° C. for 2minutes, and a final elongation step at 72° C. for 10 minutes.

To characterize the function of the Ostreococcus lucimarinus elongases,the open reading frames of the DNAs in question are cloned downstream ofthe galactose-inducible GAL1 promoter of pYES2.1/V5-His-TOPO(Invitrogen), giving rise to pOLE1 and pOLE2.

The Saccharomyces cerevisiae strain 334 is transformed with the vectorpOLE1 or pOLE2, respectively, by electroporation (1500 V). A yeast whichis transformed with the blank vector pYES2 is used as control. Thetransformed yeasts are selected on complete minimal dropout uracilmedium (CMdum) agar plates supplemented with 2% glucose. After theselection, in each case three transformants are selected for the furtherfunctional expression.

To express the OI elongases, precultures consisting of in each case 5 mlof CMdum dropout uracil liquid medium supplemented with 2% (w/v)raffinose are initially inoculated with the selected transformants andincubated for 2 days at 30° C. and 200 rpm. Then, 5 ml of CMdum (dropouturacil) liquid medium supplemented with 2% raffinose and 300 μM variousfatty acids are inoculated with the precultures to an OD₆₀₀ of 0.05.Expression is induced by the addition of 2% (w/v) galactose. Thecultures were incubated for a further 96 hours at 20° C.

Yeasts which have been transformed with the plasmids pYES2, pOLE1 andpOLE2 are analyzed as follows: The yeast cells from the main culturesare harvested by centrifugation (100×g, 5 min, 20° C.) and washed with100 mM NaHCO₃, pH 8.0 to remove residual medium and fatty acids.Starting with the yeast cell sediments, fatty acid methyl esters (FAMEs)are prepared by acid methanolysis. To this end, the cell sediments areincubated for one hour at 80° C. together with 2 ml of 1 N methanolicsulfuric acid and 2% (v/v) of dimethoxypropane. The FAMEs are extractedtwice with petroleum ether (PE). To remove nonderivatized fatty acids,the organic phases are washed in each case once with 2 ml of 100 mMNaHCO₃, pH 8.0 and 2 ml of distilled water. Thereafter, the PE phasesare dried with Na₂SO₄, evaporated under argon and taken up in 100 μl ofPE. The samples are separated on a DB-23 capillary column (30 m, 0.25mm, 0.25 μm, Agilent) in a Hewlett-Packard 6850 gas chromatographequipped with flame ionization detector. The conditions for the GLCanalysis are as follows: the oven temperature was programmed from 50° C.to 250° C. with an increment of 5° C./min and finally 10 min at 250° C.(holding).

The signals are identified by comparing the retention times withcorresponding fatty acid standards (Sigma). The methodology is describedfor example in Napier and Michaelson, 2001, Lipids. 36(8):761-766;Sayanova et al., 2001, Journal of Experimental Botany.52(360):1581-1585, Sperling et al., 2001, Arch. Biochem. Biophys.388(2):293-298 and Michaelson et al., 1998, FEBS Letters.439(3):215-218.

Activity and substrate determination of D5EIo(OI): To determine theactivity and substrate specificity of d5EIo(OI), various fatty acidswere fed (table 3). The substrates fed can be detected in large amountsin all of the transgenic yeasts. The transgenic yeasts reveal thesynthesis of novel fatty acids, the products of the d5EIo(OI) reaction.This means that the gene d5EIo(OI) was expressed functionally.

TABLE 3 Feeding of yeasts with the plasmids pYES and pYES-D5Elo(OI)Sample name/fatty acid fed Expected conversion Substrate Product pYESControl — — pYES-D5Elo(OI_GA) 20:4 20:4 → 22:4 98.0 48.5pYES-D5Elo(OI_GA) 20:4 20:4 → 22:4 62.6 32.2

FIG. 6 shows the chromatograms of the individual experiments. In FIG. 6a, yeasts transformed with pYES were analyzed without the addition offatty acids by way of control. In FIG. 1b , the pYES-transformed yeastswere fed the fatty acid 20:4Δ5,8,11,14. Here, the fed fatty acid can bedetected in large amounts. The same experiment is carried out in FIG. 6Cfor yeasts transformed with the plasmid pYES-D5EIo(OI). As opposed toFIG. 6B, it is possible to detect, in the yeasts with pYES-D5EIo(OI), anadditional fatty acid, which must be attributed to the D5EIo(OI)activity. With reference to the activity, it is possible to characterizeD5EIo(OI) as a Δ5-elongase.

Summary of the D5EIo(OI) Results:

It was possible to demonstrate in the yeast feeding experiments that thecloned gene D5EIo(OI) SEQ ID12 was expressed functionally and that ithas elongase activity. By reference of the fed fatty acid, it ispossible to characterize D5EIo(OI) as a Δ5-elongase, i.e. C20-fattyacids with a Δ5-double bond are elongated specifically.

Activity and Substrate Determination of D6EIo(OI):

To determine the activity and substrate specificity of D6EIo(OI),various fatty acids were fed (table 4). The substrates fed can bedetected in large amounts in all of the transgenic yeasts. Thetransgenic yeasts reveal the synthesis of novel fatty acids, theproducts of the D6EIo(OI) reaction. This means that the gene D6EIo(OI)was expressed functionally.

TABLE 4 Feeding/conversion of various fatty acids with D6Elo(OI) Samplename/fatty acid fed Expected conversion Substrate Product pYES Control —— pYES-D6Elo(OI_GA) γ18:3 γ18:3 → 20:3 236.5 232.1 pYES-D6Elo(OI_GA)γ18:3 γ18:3 → 20:3 111.2 126.5 pYES-D6Elo(OI_GA) 18:4 18:4 → 20:4 94.382.9 pYES-D6Elo(OI_GA) 18:4 18:4 → 20:4 73.2 68.3

FIG. 7 shows the chromatograms of the individual experiments. In FIG. 7a, yeasts transformed with pYES were analyzed without the addition offatty acids by way of control. In FIGS. 7b and 7c , the pYES-transformedyeasts were fed the fatty acid 18:3Δ6,9,12(b) and 18:4Δ6,9,12,15(c),respectively. Here, the fed fatty acids can be detected in largeamounts. The same experiment is carried out in FIGS. 7C and 7D foryeasts transformed with the plasmid pYES-D6EIo(OI). As opposed to FIGS.7B and 7C, it is possible to detect, in the yeasts with pYES-D6EIo(OI),an additional fatty acid, which must be attributed to the D6EIo(OI)activity. With reference to the activity, it is possible to characterizeD6EIo(OI) as a Δ6-elongase.

Summary of the D6EIo(OI) Results:

It was possible to demonstrate in the yeast feeding experiments that thecloned gene D6EIo(OI) SEQ ID NO: 16 was expressed functionally and thatit has elongase activity. By reference to the fed fatty acid, it ispossible to characterize D6EIo(OI) as a Δ6-elongase, i.e. C18-fattyacids with a Δ6-double bond are elongated specifically.

Example 5 Cloning and Characterization of Ostreococcus lucimarinusDesaturase Genes

By searching for conserved regions in the protein sequences with the aidof conserved motifs (His boxes, Domergue et al. 2002, Eur. J. Biochem.269, 4105-4113), it was possible to identify five sequences withcorresponding motifs in an Ostreococcus lucimarinus sequence database(genomic sequences). In a further step, the genes were characterized bymeans of sequence alignment, gene prediction and the search for codingregions. The following coding regions were found:

TABLE 5 Coding regions Name of gene SEQ ID Amino acids D4Des(OI) 6 466D5Des(OI) 8 459 D5Des_2(OI) 10 491 D6Des(OI) 14 482 D12Des(OI) 4 442D12Des_2(OI) 2 362

To characterize the function of the Ostreococcus lucimarinus desaturased6Des(OI) (=Δ6-desaturase), the open reading frame of the DNA is cloneddownstream of the galactose-inducible GAL1 promoter ofpYES2.1/V5-His-TOPO (Invitrogen), giving rise to the correspondingpYES2.1-d6EIo(OI) clone. Further desaturase genes from Ostreococcus canbe cloned accordingly.

The Saccharomyces cerevisiae strain 334 is transformed with the vectorpYES2.1-d6EIo(OI), by electroporation (1500 V). A yeast which istransformed with the blank vector pYES2 was used as control. Thetransformed yeasts were selected on complete minimal dropout uracilmedium (CMdum) agar plates supplemented with 2% glucose. After theselection, in each case three transformants were selected for thefurther functional expression.

To express the d6EIo(OI) desaturase, precultures consisting of in eachcase 5 ml of CMdum dropout uracil liquid medium supplemented with 2%(w/v) raffinose are initially inoculated with the selected transformantsand incubated for 2 days at 30° C. and 200 rpm. Then, 5 ml of CMdum(dropout uracil) liquid medium supplemented with 2% raffinose and 300 μMvarious fatty acids are inoculated with the precultures to an OD₆₀₀ of0.05. Expression is induced by the addition of 2% (w/v) galactose. Thecultures are incubated for a further 96 hours at 20° C.

In the ClustalW sequence alignment, FIGS. 2 to 5 show sequencesimilarities with other algae (Ostreococcus tauri, Thalassiosirapseudonana) for the various desaturase amino acid sequences.Surprisingly, the O. lucimarinus sequences differ markedly in theiramino acid sequence from the other algae.

TABLE 6 Sequence identities of individual desaturases Name of gene SEQID Organism Identity in % D4Des(OI) 6 O. lucimarinus 100 O. tauri 69 T.pseudonana 20 D5Des(OI) 8 D5Des_2(OI) 23 O. tauri_2 47 T. pseudonana 22D5Des_2(OI) 10 D5Des(OI) 23 O. tauri_2 14 T. pseudonana 19 D6Des(OI) 14O. lucimarinus 100 O. tauri 62 T. pseudonana 15 D12Des(OI) 4D12Des_2(OI) 51 O. tauri 82 T. pseudonana 34 D12Des_2(OI) 2 D12Des(OI)51 O. tauri 47 T. pseudonana 32

The genes are characterized as follows:

To express the desaturases in yeast cells are harvested from the maincultures by centrifugation (100×g, 5 min, 20° C.) and washed with 100 mMNaHCO₃, pH 8.0 to remove residual medium and fatty acids. The yeast cellsediments are extracted for 4 hours using chloroform/methanol (1:1). Theresulting organic phase is extracted with 0.45% NaCl, dried with Na₂SO₄and evaporated in vacuo. Applying thin-layer chromatography (horizontaltank, chloroform:methanol:acetic acid 65:35:8), the lipid extract isseparated further into the lipid classes phosphatidylcholine (PC),phosphatidylinosotol (PI), phosphatidylserine (PS),phosphatidylethanolamine (PE) and neutral lipids (NL). The variousseparated spots on the thin-layer plate are scraped off. For thegas-chromatographic analysis, fatty acid methyl esters (FAMEs) wereprepared by acid methanolysis. To this end, the cell sediments areincubated for one hour at 80° C. together with 2 ml of 1 N methanolicsulfuric acid and 2% (v/v) dimethoxypropane. The FAMEs were extractedtwice with petroleum ether (PE). To remove nonderivatized fatty acids,the organic phase is washed in each case once with 2 ml of 100 mMNaHCO₃, pH 8.0 and 2 ml of distilled water. Thereafter, the PE phasesare dried with Na₂SO₄, evaporated under argon and taken up in 100 μl ofPE. The samples are separated on a DB-23 capillary column (30 m, 0.25mm, 0.25 μm, Agilent) in a Hewlett-Packard 6850 gas chromatographequipped with flame ionization detector. The conditions for the GLCanalysis are as follows: the oven temperature is programmed from 50° C.to 250° C. with a 5° C./min increment and finally 10 min at 250° C.(holding).

The signals are identified by comparing the retention times withcorresponding fatty acid standards (Sigma). The methodology is describedfor example in Napier and Michaelson, 2001, Lipids. 36(8):761-766;Sayanova et al., 2001, Journal of Experimental Botany.52(360):1581-1585, Sperling et al., 2001, Arch. Biochem. Biophys.388(2):293-298 and Michaelson et al., 1998, FEBS Letters.439(3):215-218.

Activity and Substrate Determination of D5Des_2 (OI):

To determine the activity and substrate specificity of D5Des_2 (OI) SEQID NO: 10, various fatty acids were fed (table 7). The substrates fedcan be detected in large amounts in all of the transgenic yeasts. Thetransgenic yeasts reveal the synthesis of novel fatty acids, theproducts of the D5Des_2 (OI) reaction. This means that the gene D5Des_2(OI) was expressed functionally.

TABLE 7 Feeding/conversion of different fatty acids by D5Des(OI_2).Sample name/fatty acid fed Expected conversion Substrate Product pYESControl — — pYES-d5Des(OI_GA) w/o FS — pYES-d5Des(OI_GA) 20:3 20:3 →20:4ara 11.1 0.9

FIG. 8 shows the chromatograms of the individual experiments. In FIG. 8a, yeasts transformed with pYES were analyzed without the addition offatty acids by way of control. In FIG. 8b , the pYES-transformed yeastswere fed the fatty acid 20:3Δ8,11,14. Here, the fed fatty acid can bedetected in large amounts. The same experiment is carried out in FIG. 8Cfor yeasts transformed with the plasmid pYES-D5Des(OI_2). As opposed toFIG. 8B, it is possible to detect, in the yeasts with pYES-D5Des(OI_2),an additional fatty acid, which must be attributed to the D5Des(OI_2)activity. With reference to the activity, it is possible to characterizeD5Des(OI_2) as a Δ5-desaturase.

Summary of the D5Des_2 (OI) Results:

It was possible to demonstrate in the yeast feeding experiments that thecloned gene D5Des_2 (OI) SEQ ID NO: 10 was expressed functionally andthat it has desaturase activity. By reference to the fed fatty acid, itis possible to characterize D5Des_2 (OI) as a Δ5-desaturase, i.e.C20-fatty acids with a Δ8-double bond are dehydrogenated specifically inthe Δ5 position.

Activity and Substrate Determination of D12Des(OI):

To determine the activity and substrate specificity of D12Des(OI) SEQ IDNO: 4, various fatty acids were fed (table 8). The substrates fed can bedetected in large amounts in all of the transgenic yeasts. Thetransgenic yeasts reveal the synthesis of novel fatty acids, theproducts of the D12Des(OI) reaction. This means that the gene D12Des(OI)was expressed functionally.

TABLE 8 Feeding/conversion of different fatty acids by D12Des(OI).Sample name/fatty acid fed Expected conversion Substrate Product pYESControl — — pYES-D12Des(OI) 18:1 → 18:2 24.9 1.1 pYES-D12Des(OI) 18:1 →18:2 24.1 1.0

FIG. 9 shows the chromatograms of the individual experiments. In FIG. 9a, yeasts transformed with pYES were analyzed without the addition offatty acids by way of control. In FIG. 9b , the yeasts transformed withpYES-D12Des(OI) were analyzed. As opposed to FIG. 9a , it is possible todetect, in the yeasts with pYES-D12Des(OI), an additional fatty acid,which must be attributed to the D12Des(OI) activity. With reference tothe activity, it is possible to characterize D12Des(OI) as aΔ12-desaturase.

Summary of the D12Des(OI) Results:

It was possible to demonstrate in the yeast feeding experiments that thecloned gene D12Des(OI) SEQ ID NO: 4 was expressed functionally and thatit has desaturase activity. By reference to the fatty acid spectrum, itis possible to characterize D12Des(OI) as a Δ12-desaturase, i.e.C18-fatty acids with a Δ9-double bond are dehydrogenated specifically inthe Δ12 position.

Activity and substrate determination of D5Des(OI): To determine theactivity and substrate specificity of D5Des(OI) SEQ ID NO: 8, variousfatty acids were fed (table 9). The substrates fed can be detected inlarge amounts in all of the transgenic yeasts. The transgenic yeastsreveal the synthesis of novel fatty acids, the products of the D5Des(OI)reaction. This means that the gene D5Des(OI) was expressed functionally.

TABLE 9 Conversion of various fatty acids by D5Des(OI) Conversion SampleExpected conversion Substrate Product rate [%] d5Des(OI_febit) 20:3n-6 →20:4ara 29.4 12.4 29.6 d5Des(OI_febit) 20:3n-6 → 20:4ara 19.8 10.3 34.3d5Des(OI_febit) 20:4n-3 → 20:5 nd 1.2 >50%

FIG. 10 shows the gas-chromatographic analysis of yeast feedingexperiments. After expression of pYes-d5Des(OI_1) in yeast strain InvScwithout the addition of fatty acids (FIG. 10A), no conversion of theexisting fatty acids was detected. pYes-d5Des(OI_1) expression in yeaststrain InvSc after addition of the fatty acid 20:3n-6 (B) leads to thespecific conversion of 20:3n-6 into 20:4n-6 (arachidonic acid), andexpression of pYes-d5Des(OI_1) in yeast strain InvSc after addition ofthe fatty acid 20:4n-3 (C) leads to the specific conversion of 20:4n-3into 20:5n-3 (eicosapentaenoic acid). The specific incorporation of d5double bonds into the fed fatty acids shows the d5-desaturase activityof d5Des(OI).

Summary of the D5Des(OI) Results:

It was possible to demonstrate in the yeast feeding experiments that thecloned gene D5Des(OI) SEQ ID NO: 8 was expressed functionally and thatit has desaturase activity. By reference to the fatty acid spectrum, itis possible to characterize D5Des(OI) as a Δ5-desaturase, i.e. C20-fattyacids with a Δ8-double bond are dehydrogenated specifically in the Δ5position.

We claim:
 1. A polynucleotide comprising an expression control sequenceoperably linked to a heterologous nucleic acid sequence selected fromthe group consisting of: a) the nucleic acid sequence of SEQ ID NO: 9;b) a nucleic acid sequence encoding a polypeptide comprising the aminoacid sequence of SEQ ID NO: 10; c) a nucleic acid sequence having atleast 80% sequence identity to the nucleic acid sequence of a) or b),wherein said nucleic acid sequence encodes a polypeptide havingdesaturase activity; and d) a nucleic acid sequence encoding apolypeptide having at least 80% sequence identity to the amino acidsequence of SEQ ID NO: 10, wherein said polypeptide has desaturaseactivity.
 2. The polynucleotide of claim 1, wherein said polynucleotidefurther comprises a terminator sequence operably linked to the nucleicacid sequence.
 3. A vector comprising the polynucleotide of claim
 1. 4.A host cell comprising: a) the polynucleotide of claim 1; or b) a vectorcomprising said polynucleotide.
 5. A non-human transgenic organismcomprising: a) the polynucleotide of claim 1; or b) a vector comprisingsaid polynucleotide.
 6. The non-human transgenic organism of claim 5,wherein the organism is an animal, a plant, a multicellularmicroorganism, or a single-celled microorganism.
 7. A method for themanufacture of polyunsaturated fatty acids, comprising: a) cultivatingthe host cell of claim 4 under conditions which allow for the productionof polyunsaturated fatty acids in said host cell; and b) obtaining saidpolyunsaturated fatty acids from said host cell.
 8. A method for themanufacture of polyunsaturated fatty acids, comprising: a) cultivatingthe non-human transgenic organism of claim 5 under conditions whichallow for the production of polyunsaturated fatty acids in said hostcell; and b) obtaining said polyunsaturated fatty acids from saidnon-human transgenic organism.
 9. The method of claim 8, wherein saidpolyunsaturated fatty acid is arachidonic acid (ARA), eicosapentaenoicacid (EPA), and/or docosahexaenoic acid (DHA).
 10. A method for themanufacture of an oil, lipid, or fatty acid composition, comprising: a)cultivating the host cell of claim 4 under conditions which allow forthe production of polyunsaturated fatty acids in said host cell; b)obtaining said polyunsaturated fatty acids from said host cell; and c)formulating the polyunsaturated fatty acid as an oil, lipid, or fattyacid composition.
 11. The method of claim 10, wherein said oil, lipid,or fatty acid composition is to be used for feed, foodstuffs, cosmetics,or pharmaceuticals.
 12. A method for the manufacture of polyunsaturatedfatty acids, comprising: a) cultivating a plant comprising thepolynucleotide of claim 1 or a vector comprising said polynucleotideunder conditions which allow for the production of polyunsaturated fattyacids in said plant or seeds thereof; and b) obtaining saidpolyunsaturated fatty acids from said plant or seeds thereof.
 13. Themethod of claim 12, wherein the polyunsaturated fatty acids are obtainedfrom the seeds of said plant.
 14. The method of claim 12, comprisingobtaining an oil-, lipid- or fatty acid-composition from said plant orseeds thereof, and obtaining the polyunsaturated fatty acids from saidoil-, lipid- or fatty acid-composition.
 15. A method for the manufactureof an oil-, lipid- or fatty acid-composition, comprising: a) providing apolyunsaturated fatty acid produced by the method of claim 12; and b)formulating said polyunsaturated fatty acid as an oil-, lipid- or fattyacid-composition.
 16. A method for the manufacture of an oil-, lipid- orfatty acid-composition, comprising: a) cultivating a plant comprisingthe polynucleotide of claim 1 or a vector comprising said polynucleotideunder conditions which allow for the production of polyunsaturated fattyacids in said plant or seeds thereof; and b) obtaining an oil-, lipid-or fatty acid-composition from said plant or seeds thereof.
 17. Themethod of claim 16, wherein the oil-, lipid- or fatty acid-compositionis obtained from the seeds of said plant.
 18. An oil-, lipid- or fattyacid-composition obtained by the method of claim 16, wherein said oil-,lipid- or fatty acid-composition comprises a non-naturally occurringcomposition and content of polyunsaturated fatty acids.
 19. The oil-,lipid- or fatty acid-composition of claim 18, wherein said oil-, lipid-or fatty acid-composition comprises arachidonic acid (ARA),eicosapentaenoic acid (EPA), and/or docosahexaenoic acid (DHA).
 20. Amethod for the production of feed, foodstuffs, cosmetics orpharmaceuticals, comprising: a) obtaining an oil-, lipid- or fattyacid-composition produced by the method of claim 16; and b) processingsaid oil-, lipid- or fatty acid-composition to produce feed, foodstuffs,cosmetics or pharmaceuticals.